Subtilase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to subtilase variants suitable for use in, e.g., cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions. The present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to subtilase variants suitable for use in,e.g., cleaning or detergent compositions, such as laundry detergentcompositions and dish wash compositions, including automatic dish washcompositions. The present invention also relates to isolated DNAsequences encoding the variants, expression vectors, host cells, andmethods for producing and using the variants of the invention.

Description of the Related Art

In the detergent industry, enzymes have for many decades beenimplemented in washing formulations. Enzymes used in such formulationscomprise amylases, cellulases, lipases, mannosidases, and proteases, aswell as other enzymes or mixtures thereof. Commercially the mostimportant enzymes are proteases.

An increasing number of commercially used proteases are proteinengineered variants of naturally occurring wild type proteasesEverlase®, Relase®, Ovozyme®, Polarzyme®, Liquanase®, Liquanase Ultra®and Kannase® (Novozymes A/S), Purafast®, Purafect OXP®, FN3®, FN4® andExcellase® (Genencor International, Inc.). Further, a number of variantsare described in the art, such as in WO1996/034946, WO 2004/041979 andWO2000/037599 (Novozymes A/S) which describes subtilase variantsexhibiting alterations relative to the parent subtilase in, e.g., washperformance, thermal stability, storage stability or catalytic activity.The variants are suitable for use in, e.g., cleaning or detergentcompositions.

A number of subtilase variants have been described many of which haveprovided improved activity, stability, and solubility in differentdetergents

However, various factors make further improvement of the proteasesadvantageous. Washing conditions such as temperature and pH change overtime and many stains are still difficult to completely remove underconventional washing conditions. Despite intensive research in proteasedevelopment there remains a need for proteases that have improved washperformance compared to the parent subtilase.

SUMMARY OF THE INVENTION

The present invention relates to subtilase variants having proteaseactivity and comprising a set of alterations selected from the groupconsisting of:

(a) X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and one or moresubstitutions selected from the group consisting of X59D (e.g., Q59D),X62D (e.g., N62D), X76D (e.g., N76D), X104T (e.g., V104T), X120D (e.g.,H120D), X133P (e.g., A133P), X141N (e.g., S141N), X156D (e.g., S156D),X163G (e.g., S163G), X209W (e.g., Y209W), X228V (e.g., A228V), X230V(e.g., A230V), X238E (e.g., N238E), X261D (e.g., N261D), and X262E(e.g., L262E);

(b)*99aE and one or more substitutions selected from the groupconsisting of X21D (e.g., L21D), X59D (e.g., Q59D), X101H (e.g., S101H),X120D (e.g., H120D), X156D (e.g., S156D), X163G (e.g., S163G), X194P(e.g., A194P), X195E (e.g., G195E), X209W (e.g., Y209W), X238E (e.g.,N238E), X256D (e.g., N256D), X261D (e.g., N261D), and X262E (e.g.,L262E);

(c) X62D (e.g., N62D) and one or more substitutions selected from thegroup consisting of X101H (e.g., S101H), X104T (e.g., V104T), X156D(e.g., S156D), X163G (e.g., S163G), X170S (e.g., R170S), X170L (e.g.,R170L), X209W (e.g., Y209W), X238E (e.g., N238E), X245R (e.g. Q245R) andX262E (e.g., L262E);

(d) X62D+X245R+X248D (e.g., N62D+Q245R+N248D) and one or moresubstitutions selected from the group consisting of X156D (e.g., S156D),X163G (e.g., S163G), X163K (e.g., S163K), X170S (e.g., R170S), X209W(e.g., Y209W), and X262E (e.g., L262E);

(e) X170L, X170N or X170S (e.g., R170L, R170N or R170S) and one or moresubstitutions selected from the group consisting of X57P (e.g., S57P),X167A (e.g., Y167A), X172E (e.g. A172E), X206E (e.g., Q206E),

(f) X99D (e.g., S99D) and one or more substitutions selected from thegroup consisting of *97aN, *98aA, X98T (e.g., A98T), X261D (e.g.,N261D), and X262Q (e.g., L262Q); wherein the positions correspond to thepositions of the polypeptide of SEQ ID NO: 2.

The present invention further relates to polynucleotides encoding thesubtilase variants and methods for obtaining a subtilase variant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an alignment of the amino acid sequences of subtilisin 309(SEQ ID NO: 1) and subtilisin BPN′ (SEQ ID NO: 2), using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453).

DEFINITIONS

The term “allelic variant” means any of two or more alternative forms ofa gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The term “cDNA” means a DNA molecule that can be prepared by reversetranscription from a mature, spliced, mRNA molecule obtained from aeukaryotic or prokaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps, including splicing, before appearing as mature spliced mRNA.

The term “coding sequence” means a polynucleotide, which directlyspecifies the amino acid sequence of a variant. The boundaries of thecoding sequence are generally determined by an open reading frame, whichbegins with a start codon such as ATG, GTG or TTG and ends with a stopcodon such as TAA, TAG, or TGA. The coding sequence may be a genomicDNA, cDNA, synthetic DNA, or a combination thereof.

The term “control sequences” means nucleic acid sequences necessary forexpression of a polynucleotide encoding a variant of the presentinvention. Each control sequence may be native (i.e., from the samegene) or foreign (i.e., from a different gene) to the polynucleotideencoding the variant or native or foreign to each other. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the polynucleotide encodinga variant.

The term “detergent component” is defined herein to mean the types ofchemicals which can be used in detergent compositions. Examples ofdetergent components are surfactants, hydrotropes, builders,co-builders, chelators or chelating agents, bleaching system or bleachcomponents, polymers, fabric hueing agents, fabric conditioners, foamboosters, suds suppressors, dispersants, dye transfer inhibitors,fluorescent whitening agents, perfume, optical brighteners,bactericides, fungicides, soil suspending agents, soil release polymers,anti-redeposition agents, enzyme inhibitors or stabilizers, enzymeactivators, antioxidants, and solubilizers. The detergent compositionmay comprise of one or more of any type of detergent component.

The term “detergent composition”, includes unless otherwise indicated,granular or powder-form all-purpose or heavy-duty washing agents,especially cleaning detergents; liquid, gel or paste-form all-purposewashing agents, especially the so-called heavy-duty liquid (HDL) types;liquid fine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, including antibacterial hand-wash types, cleaningbars, soap bars, mouthwashes, denture cleaners, car or carpet shampoos,bathroom cleaners; hair shampoos and hair-rinses; shower gels, foambaths; metal cleaners; as well as cleaning auxiliaries such as bleachadditives and “stain-stick” or pre-treat types. The terms “detergentcomposition” and “detergent formulation” are used in reference tomixtures which are intended for use in a wash medium for the cleaning ofsoiled objects. In some embodiments, the term is used in reference tolaundering fabrics and/or garments (e.g., “laundry detergents”). Inalternative embodiments, the term refers to other detergents, such asthose used to clean dishes, cutlery, etc. (e.g., “dishwashingdetergents”). The term “detergent composition” is not intended to belimited to compositions that contain surfactants. It is intended that inaddition to the variants according to the invention, the termencompasses detergents that may contain, e.g., surfactants, builders,chelators or chelating agents, bleach system or bleach components,polymers, fabric conditioners, foam boosters, suds suppressors, dyes,perfume, tannish inhibitors, optical brighteners, bactericides,fungicides, soil suspending agents, anticorrosion agents, enzymeinhibitors or stabilizers, enzyme activators, transferase(s), hydrolyticenzymes, oxido reductases, bluing agents and fluorescent dyes,antioxidants, and solubilizers.

In addition to containing a subtilase variant of the invention, thedetergent formulation may contain one or more additional enzymes (suchas amylases, catalases, cellulases (e.g., endoglucanases), cutinases,haloperoxygenases, lipases, mannanases, pectinases, pectin lyases,peroxidases, proteases, xanthanases, and xyloglucanases, or any mixturethereof), and/or components such as surfactants, builders, chelators orchelating agents, bleach system or bleach components, polymers, fabricconditioners, foam boosters, suds suppressors, dyes, perfume, tannishinhibitors, optical brighteners, bactericides, fungicides, soilsuspending agents, anti-corrosion agents, enzyme inhibitors orstabilizers, enzyme activators, transferase(s), hydrolytic enzymes,oxidoreductases, bluing agents and fluorescent dyes, antioxidants, andsolubilizers.

The term “dish wash” refers to all forms of washing dishes, e.g., byhand or automatic dish wash. Washing dishes includes, but is not limitedto, the cleaning of all forms of crockery such as plates, cups, glasses,bowls, all forms of cutlery such as spoons, knives, forks and servingutensils as well as ceramics, plastics such as melamine, metals, china,glass and acrylics.

The term “dish washing composition” refers to all forms of compositionsfor cleaning hard surfaces. The present term is not restricted to anyparticular type of dish wash composition or any particular detergent.

The term “expression” includes any step involved in the production of avariant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “expression vector” means a linear or circular DNA moleculethat comprises a polynucleotide encoding a variant and is operablylinked to control sequences that provide for its expression.

The term “hard surface cleaning” is defined herein as cleaning of hardsurfaces wherein hard surfaces may include floors, tables, walls, roofsetc. as well as surfaces of hard objects such as cars (car wash) anddishes (dish wash). Dishwashing includes but are not limited to cleaningof plates, cups, glasses, bowls, and cutlery such as spoons, knives,forks, serving utensils, ceramics, plastics such as melamine, metals,china, glass and acrylics.

The term “host cell” means any cell type that is susceptible totransformation, transfection, transduction, or the like with a nucleicacid construct or expression vector comprising a polynucleotide of thepresent invention. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication.

The term “improved property” means a characteristic associated with asubtilase variant that is improved compared to the parent subtilase.Such improved properties include, but are not limited to, washperformance, protease activity, thermal activity profile,thermostability, pH activity profile, pH stability, substrate/cofactorspecificity, improved surface properties, substrate specificity, productspecificity, increased stability, improved stability under storageconditions, and chemical stability.

The term “stability” includes storage stability and stability duringuse, e.g., during a wash process and reflects the stability of thesubtilase variant according to the invention as a function of time,e.g., how much activity is retained when the subtilase variant is keptin solution in particular in a detergent solution. The stability isinfluenced by many factors, e.g., pH, temperature, detergentcomposition, e.g., amount of builder, surfactants etc. The term“improved stability” or “increased stability” is defined herein as avariant subtilase displaying an increased stability in solution,relative to the stability of the parent subtilase. The terms “improvedstability” and “increased stability” includes “improved chemicalstability”, “detergent stability” or “improved detergent stability.

The term “improved chemical stability” is defined herein as a variantsubtilase displaying retention of enzymatic activity after a period ofincubation in the presence of a chemical or chemicals, either naturallyoccurring or synthetic, which reduces the enzymatic activity of theparent enzyme. Improved chemical stability may also result in variantsbeing more able to catalyze a reaction in the presence of suchchemicals. In a particular aspect of the invention the improved chemicalstability is an improved stability in a detergent, in particular in aliquid detergent. The term “detergent stability” or “improved detergentstability is in particular an improved stability of the proteaseactivity when a subtilase variant of the present invention is mixed intoa liquid detergent formulation, and then stored at a temperature between15 and 50° C., e.g., 20° C., 30° C. or 40° C.

The term “improved thermal activity” means a variant displaying analtered temperature-dependent activity profile at a specific temperaturerelative to the temperature-dependent activity profile of the parent.The thermal activity value provides a measure of the variant'sefficiency in enhancing catalysis of a hydrolysis reaction over a rangeof temperatures. A more thermo active variant will lead to an increasein enhancing the rate of hydrolysis of a substrate by an enzymecomposition thereby decreasing the time required and/or decreasing theenzyme concentration required for activity. Alternatively, a variantwith a reduced thermal activity will enhance an enzymatic reaction at atemperature lower than the temperature optimum of the parent defined bythe temperature-dependent activity profile of the parent.

The term “improved wash performance” is defined herein as a subtilasevariant according to the invention displaying an improved washperformance relative to the wash performance of the parent protease,e.g., by increased stain removal. The term “wash performance” includeswash performance in laundry but also, e.g., in dish wash. The washperformance may be quantified as described under the definition of “washperformance” herein.

The term “isolated” means a substance in a form or environment whichdoes not occur in nature. Non-limiting examples of isolated substancesinclude (1) any non-naturally occurring substance, (2) any substanceincluding, but not limited to, any enzyme, variant, nucleic acid,protein, peptide or cofactor, that is at least partially removed fromone or more or all of the naturally occurring constituents with which itis associated in nature; (3) any substance modified by the hand of manrelative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

The term “laundering” relates to both household laundering andindustrial laundering and means a process of treating textiles and/orfabrics with a solution containing a detergent composition. Thelaundering process can for example be carried out using, e.g., ahousehold or an industrial washing machine or can be carried out byhand.

The term “mature polypeptide” means a polypeptide in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, autocatalytic activation etc. In one aspect, the maturepolypeptide is amino acids 1 to 269 of SEQ ID NO: 1 and 1 to 275 of SEQID NO: 2. It is known in the art that a host cell may produce a mixtureof two of more different mature polypeptides (i.e., with a differentC-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide.

The term “mature polypeptide coding sequence” means a polynucleotidethat encodes a mature polypeptide having protease activity.

The term “mutant” means a polynucleotide encoding a variant.

The term “nucleic acid construct” means a nucleic acid molecule, eithersingle- or double-stranded, which is isolated from a naturally occurringgene or is modified to contain segments of nucleic acids in a mannerthat would not otherwise exist in nature or which is synthetic, whichcomprises one or more control sequences.

The term “operably linked” means a configuration in which a controlsequence is placed at an appropriate position relative to the codingsequence of a polynucleotide such that the control sequence directsexpression of the coding sequence.

The term “parent” means a protease to which an alteration is made toproduce the enzyme variants of the present invention. It will beunderstood that in the present context the expression “having identicalamino acid sequence” relates to 100% sequence identity. In a particularembodiment the parent is a protease with at least 60% identity, such asat least 65%, at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity toa polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

The term “protease” is defined herein as an enzyme that hydrolyzespeptide bonds. It includes any enzyme belonging to the EC 3.4 enzymegroup (including each of the thirteen subclasses thereof). The EC numberrefers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, SanDiego, Calif., including supplements 1-5 published in Eur. J. Biochem.1223: 1-5 (1994); Eur. J. Biochem. 232: 1-6 (1995); Eur. J. Biochem.237: 1-5 (1996); Eur. J. Biochem. 250: 1-6 (1997); and Eur. J. Biochem.264: 610-650 (1999); respectively. The most widely used proteases in thedetergent industry such as laundry and dish wash are the serineproteases or serine peptidases which is a subgroup of proteasescharacterised by having a serine in the active site, which forms acovalent adduct with the substrate. Further the subtilases (and theserine proteases) are characterized by having two active site amino acidresidues apart from the serine, namely a histidine residue and anaspartic acid residue. Subtilase refer to a sub-group of serine proteaseaccording to Siezen et al., 1991, Protein Engng. 4: 719-737 and Siezenet al., 1997, Protein Science 6: 501-523. The subtilases may be dividedinto 6 sub-divisions, i.e., the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family. The term “protease activity”means a proteolytic activity (EC 3.4). Proteases usably in detergentsare mainly endopeptidases (EC 3.4.21). There are several proteaseactivity types: The three main activity types are: trypsin-like wherethere is cleavage of amide substrates following Arg or Lys at P1,chymotrypsin-like where cleavage occurs following one of the hydrophobicamino acids at P1, and elastase-like with cleavage following an Ala atP1. For purposes of the present invention, protease activity isdetermined according to the Suc-AAPF-pNA activity assay, as described inthe Materials and Methods section below. In one aspect, the subtilasevariants of the present invention have at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 100% of the enzyme activity of the maturepolypeptide of the parent enzyme. In one particular aspect the subtilasevariants of the present invention have at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 100% of the enzyme activity of a polypeptide ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

The term “protease activity” means a proteolytic activity (EC 3.4).Proteases of the invention are endopeptidases (EC 3.4.21). There areseveral protease activity types: The three main activity types are:trypsin-like where there is cleavage of amide substrates following Argor Lys at P1, chymotrypsin-like where cleavage occurs following one ofthe hydrophobic amino acids at P1, and elastase-like with cleavagefollowing an Ala at P1. For purposes of the present invention, proteaseactivity is determined according to the procedure described in“Materials and Methods” below. The subtilase variants of the presentinvention preferably have at least 20%, e.g., at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, and at least 100% of the protease activity of a polypeptide of SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “sequence identity”.For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

The different stringency conditions are defined as follows.

The term “very low stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 35% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 1×SSC, 0.2% SDS at 60° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 1×SSC, 0.2% SDS at 65° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 0.5×SSC, 0.2% SDS at 65° C.

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 0.3×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 35% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 0.15×SSC, 0.2% SDS at 65° C.

The term “substantially pure variant” means a preparation that containsat most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%,at most 2%, at most 1%, and at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated.Preferably, the variant is at least 92% pure, e.g., at least 94% pure,at least 95% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, at least 99.5% pure, and 100% pure by weight of thetotal polypeptide material present in the preparation. The variants ofthe present invention are preferably in a substantially pure form. Thiscan be accomplished, for example, by preparing the variant by well-knownrecombinant methods or by classical purification methods.

The term “substantially pure polynucleotide” means a polynucleotidepreparation free of other extraneous or unwanted nucleotides and in aform suitable for use within genetically engineered polypeptideproduction systems. Thus, a substantially pure polynucleotide containsat most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%,at most 2%, at most 1%, and at most 0.5% by weight of otherpolynucleotide material with which it is natively or recombinantlyassociated. A substantially pure polynucleotide may, however, includenaturally occurring 5′- and 3′-untranslated regions, such as promotersand terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, e.g., at least 92% pure, at least94% pure, at least 95% pure, at least 96% pure, at least 97% pure, atleast 98% pure, at least 99% pure, and at least 99.5% pure by weight.The polynucleotides of the present invention are preferably in asubstantially pure form.

The term “textile” refers to woven fabrics, as well as staple fibers andfilaments suitable for conversion to or use as yarns, woven, knit, andnon-woven fabrics. The term encompasses yarns made from natural, as wellas synthetic (e.g., manufactured) fibers. The term, “textile materials”is a general term for fibers, yarn intermediates, yarn, fabrics, andproducts made from fabrics (e.g., garments and other articles).

The term “non-fabric detergent compositions” include non-textile surfacedetergent compositions, including but not limited to compositions forhard surface cleaning, such as dishwashing detergent compositions, oraldetergent compositions, denture detergent compositions, and personalcleansing compositions.

The term “effective amount of enzyme” refers to the quantity of enzymenecessary to achieve the enzymatic activity required in the specificapplication, e.g., in a defined detergent composition. Such effectiveamounts are readily ascertained by one of ordinary skill in the art andare based on many factors, such as the particular enzyme used, thecleaning application, the specific composition of the detergentcomposition, and whether a liquid or dry (e.g., granular, bar)composition is required, and the like. The term “effective amount” of aprotease variant refers to the quantity of protease variant describedhereinbefore that achieves a desired level of enzymatic activity, e.g.,in a defined detergent composition.

The term “water hardness” or “degree of hardness” or “dH” or “° dH” asused herein refers to German degrees of hardness. One degree is definedas 10 milligrams of calcium oxide per litre of water.

The term “relevant washing conditions” is used herein to indicate theconditions, particularly washing temperature, time, washing mechanics,detergent concentration, type of detergent and water hardness, actuallyused in households in a detergent market segment.

The term “adjunct materials” means any liquid, solid or gaseous materialselected for the particular type of detergent composition desired andthe form of the product (e.g., liquid, granule, powder, bar, paste,spray, tablet, gel, or foam composition), which materials are alsopreferably compatible with the protease variant enzyme used in thecomposition. In some embodiments, granular compositions are in “compact”form, while in other embodiments, the liquid compositions are in a“concentrated” form.

The term “stain removing enzyme” as used herein, describes an enzymethat aids the removal of a stain or soil from a fabric or a hardsurface. Stain removing enzymes act on specific substrates, e.g.,protease on protein, amylase on starch, lipase and cutinase on lipids(fats and oils), pectinase on pectin and hemicellulases onhemicellulose. Stains are often depositions of complex mixtures ofdifferent components which either results in a local discolouration ofthe material by itself or which leaves a sticky surface on the objectwhich may attract soils dissolved in the washing liquor therebyresulting in discolouration of the stained area. When an enzyme acts onits specific substrate present in a stain the enzyme degrades orpartially degrades its substrate thereby aiding the removal of soils andstain components associated with the substrate during the washingprocess. For example, when a protease acts on a grass stain it degradesthe protein components in the grass and allows the green/brown colour tobe released during washing.

The term “reduced amount” means in this context that the amount of thecomponent is smaller than the amount which would be used in a referenceprocess under otherwise the same conditions. In a preferred embodimentthe amount is reduced by, e.g., at least 5%, such as at least 10%, atleast 15%, at least 20% or as otherwise herein described.

The term “low detergent concentration” system includes detergents whereless than about 800 ppm of detergent components is present in the washwater. Asian, e.g., Japanese detergents are typically considered lowdetergent concentration systems.

The term “medium detergent concentration” system includes detergentswherein between about 800 ppm and about 2000 ppm of detergent componentsis present in the wash water. North American detergents are generallyconsidered to be medium detergent concentration systems.

The term “high detergent concentration” system includes detergentswherein greater than about 2000 ppm of detergent components is presentin the wash water. European detergents are generally considered to behigh detergent concentration systems.

The term “variant” means a polypeptide having protease activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at three or more (e.g., several) positions. A substitutionmeans replacement of the amino acid occupying a position with adifferent amino acid;

a deletion means removal of the amino acid occupying a position; and aninsertion means adding one or more (e.g., several) amino acids, e.g., 1,2, 3, 4 or 5 amino acids adjacent to and immediately following the aminoacid occupying a position. The term subtilase variant means a variant ofa subtilase parent, i.e., a subtilase variant is a subtilase whichcomprises alterations i.e., a substitution, insertion, and/or deletion,at three or more (e.g., several) positions compared to the parentsubtilase.

The term “wash performance” is used as an enzyme's ability to removestains present on the object to be cleaned during, e.g., wash, such aslaundry or hard surface cleaning. The improvement in the washperformance may be quantified by calculating the so-called intensityvalue (Int) defined in the AMSA assay, as described in Example 2.

The term “wild-type subtilase” means a protease expressed by a naturallyoccurring organism, such as a bacterium, archaea, yeast, fungus, plantor animal found in nature. An example of a wild-type subtilase issubtilisin BPN′, i.e., amino acids 1 to 275 of SEQ ID NO: 2.

DETAILED DESCRIPTION OF THE INVENTION Conventions for Designation ofVariants

For purposes of the present invention, subtilisin BPN′ (the sequence ofamino acids 1-275 of SEQ ID NO: 2 (Siezen et al., 1991, Protein Eng. 4:719-737)) is used to determine the corresponding amino acid residue inanother protease. The amino acid sequence of another protease is alignedwith the mature polypeptide disclosed in SEQ ID NO: 2, and based on thealignment, the amino acid position number corresponding to any aminoacid residue in the polypeptide disclosed in SEQ ID NO: 2 is determinedusing the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol. Biol. 48: 443-453) as implemented in the Needle program of theEMBOSS package (EMBOSS: The European Molecular Biology Open SoftwareSuite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version5.0.0 or later. The parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

Identification of the corresponding amino acid residue in anotherprotease can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26:1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of histidine at position 226 with alanine is designated as“His226Ala” or “H226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Val205Ile+Leu217Asp” or “V205I+L217D”, representingsubstitutions at positions 205 and 217 of valine (V) with isoleucine (I)and leucine (L) with aspartic acid (D), respectively. The amino acidsproceeding the position i.e. the amino acid substituted with another isin the above the amino acids present in subtilisin 309 (SEQ ID NO 1) ata position corresponding to the position of BPN′ (SEQ ID NO 2). Asdescribed in “parent protease” various parent proteases are suitable formaking the variants disclosed. It will be clear to the skilled artisanthat the amino acid substituted i.e. the preceding amino acid may bedifferent for those indicated above. This could also be shown by an Xrepresenting any amino acid. An “X” preceding a position means that anyoriginal amino acid at the position may be substituted. For example, X9Emeans that any amino acid residue at position 9 other than E issubstituted with E; X76D means that any amino acid residue at position76 other than D is substituted with D; and X262E means that any aminoacid residue at position 262 other than E is substituted with E.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser103*”or “G195*+S103*”.

Insertions: The insertion of an additional amino acid residue such as,e.g., a lysine after G195 may be indicated by: Gly195GlyLys or G195GK.Alternatively insertion of an additional amino acid residue such aslysine after G195 may be indicated by: *195aK. When more than one aminoacid residue is inserted, such as, e.g., a Lys and Ala after G195 thismay be indicated as: Gly195GlyLysAla or G195GKA. In such cases, theinserted amino acid residue(s) may also be numbered by the addition oflower case letters to the position number of the amino acid residuepreceding the inserted amino acid residue(s), in this example: *195aK*195bA. In the above example, the sequences 194 to 196 would thus be:

194 195 196 Subtilisin 309 A - G - L 194 195 195a 195b 196 Variant A -G - K - A - L

In cases where a substitution and an insertion occur at the sameposition this may be indicated as S99SD+S99A or in short S99AD. The samemodification may also be indicated as S99A+*99aD.

In cases where an amino acid residue identical to the existing aminoacid residue is inserted, it is clear that degeneracy in thenomenclature arises. If for example a glycine is inserted after theglycine in the above example this would be indicated by G195GG or*195GaG. The same actual change could just as well be indicated asA194AG or *194aG for the change from:

194 195 196 Subtilisin 309 A - G - L to: 194 195 195a 196 Variant A -G - G - L 194 194a 195 196

Such instances will be apparent to the skilled person and the indicationG195GG and corresponding indications for this type of insertions arethus meant to comprise such equivalent degenerate indications.

Multiple alterations: Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.Alternatively multiple alterations may be separated by space or a comma,e.g., R170Y G195E or R170Y, G195E respectively.

Different alterations: Where different alterations can be introduced ata position, the different alterations may be separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants: “Tyr167Gly+Arg170Gly”,“Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Alternatively different alterations or optional substitutions may beindicated in brackets, e.g., Arg170[Tyr, Gly] or, Arg170{Tyr, Gly} or inshort R170 [Y,G] or R170 {Y, G}.

In an embodiment, the subtilase variants comprise X167A+R170S+A194P(e.g., Y167A+R170S+A194P) and one or more substitutions selected fromthe group consisting of X59D (e.g., Q59D), X62D (e.g., N62D), (X76D(e.g., N76D), X104T (e.g., V104T), X120D (e.g., H120D), X133P (e.g.A133P), X141N (e.g. S141N), X156D (e.g., S156D), X163G (e.g., S163G),X209W (e.g., Y209W), X228V (e.g. A228V), X230V (e.g. A230V), X238E(e.g., N238E), X261D (e.g., N261D), and X262E (e.g., L262E); andoptionally may further comprise one or alterations selected from thegroup consisting of X3T (e.g., S3T), X4I (e.g., V4I), X9C (e.g., S9C),X9D (e.g., S9D), X9E (e.g., S9E), X9Q (e.g., S9Q), X14T (e.g., A15T),X24G (e.g., S24G), X24R (e.g., S24R), X27R (e.g., K27R), *36D, X43A(e.g., N43A), X430 (e.g., N43C), X43L (e.g., N43L), X43R (e.g., N43R),X43W (e.g., N43W), X68A (e.g., V68A), X72A (e.g., I72A), X72V (e.g.,I72V), X76D (e.g., N76D), X78D (e.g., S78D), X87R (e.g., N87R), X87S(e.g., N87S), *97E, X98S (e.g., A98S), X99A (e.g., S99A), X99D (e.g.,S99D), X99A (e.g., S99A), X99D (e.g., S99D), X99E (e.g., S99E), X99G(e.g., S99G), *99aD, X101D (e.g., S101D), X101E (e.g., S101E), X101G(e.g., S101G), X101I (e.g., S101I), X101K (e.g., S101K), X101L (e.g.,S101L), X101M (e.g., S101M), X101N (e.g., S101N), X101R (e.g., S101R),X103A (e.g., S103A), X104F (e.g., V104F), X104I (e.g., V104I), X104N(e.g., V104N), X104Y (e.g., V104Y), X106A (e.g., S106A), X114V (e.g.,A114V), X115T (e.g., G115T), X115W (e.g., G115W), X118R (e.g., G118R),X118V (e.g., G118V), X120D (e.g., H120D), X120I (e.g., H120I), X120N(e.g., H120N), X120T (e.g., H120T), X120V (e.g., H120V), X123S (e.g.,N123S), X128A (e.g., S128A), X128L (e.g., S128L), X128S (e.g., S128S),X129D (e.g., P129D), X129N (e.g., P129N), X129Q (e.g., P129Q), X130A(e.g., S130A), X147W (e.g., V147W), X149C (e.g., V149C), X149N (e.g.,V149N), X158E (e.g., A158E), X160D (e.g., G160D, X160P (e.g., G160P),X161C (e.g., S161C), X161E (e.g., S161E), X162L (e.g., I162L), X163A(e.g., S163A), X163D (e.g., S163D), X182C (e.g., Q182C), X182E (e.g.,Q182E), X185C (e.g., N185C), X185E (e.g., N185E), X188C (e.g., S188C),X188D (e.g., S188D), X188E (e.g., S188E), X191N (e.g., Q191N), X195E(e.g., G195E), X199M (e.g., V199M), X204D (e.g., N204D), X204V (e.g.,N204V), X205I (e.g., V205I), X206C (e.g., Q206C), X206E (e.g., Q206E),X206I (e.g., Q206I), X206K (e.g., Q206K), X206L (e.g., Q206L), X206T(e.g., Q206T), X206V (e.g., Q206V), X206W (e.g., Q206W), X209W (e.g.,Y209W), X212A (e.g., S212A), X212D (e.g., S212D), X212G (e.g., S212G),X212N (e.g., S212N), X216I (e.g., S216I), X216T (e.g., S216T), X216V(e.g., S216V), X217C (e.g., L217C), X217D (e.g., L217D), X217E (e.g.,L217E), X217M (e.g., L217M), X217Q (e.g., L217Q), X217Y (e.g., L217Y),X218D (e.g., N218D), X218E (e.g., N218E), X218T (e.g., N218T), X222C(e.g., M222C), X222R (e.g., M222R), X222S (e.g., M222S), X225A (e.g.,P225A), X232V (e.g., A232V), X235L (e.g., K235L), X236H (e.g., Q236H),X245K (e.g., Q245K), X245R (e.g., Q245R), X252K (e.g., N252K), X255C(e.g., T255C), X255E (e.g., T255E), X256A (e.g., S256A), X256C (e.g.,S256C), X256D (e.g., S256D), X256V (e.g., S256V), X256Y (e.g., S256Y),X259D (e.g., S259D), X260E (e.g., T260E), X260P (e.g., T260P), X261C(e.g., N261C), X261E (e.g., N261E), X261F (e.g., N261F), X261L (e.g.,N261L), X261M (e.g., N261M), X261V (e.g., N261V), X261W (e.g., N261W),X261Y (e.g., N261Y), X262C (e.g., L262C), X262E (e.g., L262E), X262Q(e.g., L262Q), and X274A (e.g., T274A), wherein each positioncorresponds to the position of the polypeptide of SEQ ID NO: 2.

In another embodiment, the subtilase variants comprise *99aE and one ormore substitutions selected from the group consisting of X21D (e.g.,L21D), X59D (e.g., Q59D), X101H (e.g., S101H), X120D (e.g., H120D),X156D (e.g., S156D), X163G (e.g., S163G), X194P (e.g., A194P), X195E(e.g., G195E), X209W (e.g., Y209W), X238E (e.g., N238E), X256D (e.g.N256D), X261D (e.g., N261D), and X262E (e.g., L262E); and optionally mayfurther comprise one or more alterations selected from the groupconsisting of X3T (e.g., S3T), X4I (e.g., V4I), X9C (e.g., S9C), X9D(e.g., S9D), X9E (e.g., S9E), X9Q (e.g., S9Q), X14T (e.g., A15T), X24G(e.g., S24G), X24R (e.g., S24R), X27R (e.g., K27R), *36D, X43A (e.g.,N43A), X43C (e.g., N43C), X43L (e.g., N43L), X43R (e.g., N43R), X43W(e.g., N43W), X68A (e.g., V68A), X72A (e.g., I72A), X72V (e.g., I72V),X76D (e.g., N76D), X78D (e.g., S78D), X87R (e.g., N87R), X87S (e.g.,N87S), *97E, X98S (e.g., A98S), X99A (e.g., S99A), X99D (e.g., S99D),X99A (e.g., S99A), X99D (e.g., S99D), X99E (e.g., S99E), X99G (e.g.,S99G), X101D (e.g., S101D), X101E (e.g., S101E), X101G (e.g., S101G),X101I (e.g., S101I), X101K (e.g., S101K), X101L (e.g., S101L), X101M(e.g., S101M), X101N (e.g., S101N), X101R (e.g., S101R), X103A (e.g.,S103A), X104F (e.g., V104F), X104I (e.g., V104I), X104N (e.g., V104N),X104Y (e.g., V104Y), X106A (e.g., S106A), X114V (e.g., A114V), X115T(e.g., G115T), X115W (e.g., G115W), X118R (e.g., G118R), X118V (e.g.,G118V), X120D (e.g., H120D), X120I (e.g., H120I), X120N (e.g., H120N),X120T (e.g., H120T), X120V (e.g., H120V), X123S (e.g., N123S), X128A(e.g., S128A), X128L (e.g., S128L), X128S (e.g., S128S), X129D (e.g.,P129D), X129N (e.g., P129N), X129Q (e.g., P129Q), X130A (e.g., S130A),X147W (e.g., V147W), X149C (e.g., V149C), X149N (e.g., V149N), X158E(e.g., A158E), X160D (e.g., G160D, X160P (e.g., G160P), X161C (e.g.,S161C), X161E (e.g., S161E), X162L (e.g., I162L), X163A (e.g., S163A),X163D (e.g., S163D), X167A (e.g., Y167A), X170S (e.g., R170S), X182C(e.g., Q182C), X182E (e.g., Q182E), X185C (e.g., N185C), X185E (e.g.,N185E), X188C (e.g., S188C), X188D (e.g., S188D), X188E (e.g., S188E),X191N (e.g., Q191N), X194P (e.g., A194P), X195E (e.g., G195E), X199M(e.g., V199M), X204D (e.g., N204D), X204V (e.g., N204V), X205I (e.g.,V205I), X206C (e.g., Q206C), X206E (e.g., Q206E), X206I (e.g., Q206I),X206K (e.g., Q206K), X206L (e.g., Q206L), X206T (e.g., Q206T), X206V(e.g., Q206V), X206W (e.g., Q206W), X209W (e.g., Y209W), X212A (e.g.,S212A), X212D (e.g., S212D), X212G (e.g., S212G), X212N (e.g., S212N),X216I (e.g., S216I), X216T (e.g., S216T), X216V (e.g., S216V), X217C(e.g., L217C), X217D (e.g., L217D), X217E (e.g., L217E), X217M (e.g.,L217M), X217Q (e.g., L217Q), X217Y (e.g., L217Y), X218D (e.g., N218D),X218E (e.g., N218E), X218T (e.g., N218T), X222C (e.g., M222C), X222R(e.g., M222R), X222S (e.g., M222S), X225A (e.g., P225A), X232V (e.g.,A232V), X235L (e.g., K235L), X236H (e.g., Q236H), X245K (e.g., Q245K),X245R (e.g., Q245R), X252K (e.g., N252K), X255C (e.g., T255C), X255E(e.g., T255E), X256A (e.g., S256A), X256C (e.g., S256C), X256D (e.g.,S256D), X256V (e.g., S256V), X256Y (e.g., S256Y), X259D (e.g., S259D),X260E (e.g., T260E), X260P (e.g., T260P), X261C (e.g., N261C), X261E(e.g., N261E), X261F (e.g., N261F), X261L (e.g., N261L), X261M (e.g.,N261M), X261V (e.g., N261V), X261W (e.g., N261W), X261Y (e.g., N261Y),X262C (e.g., L262C), X262E (e.g., L262E), X262Q (e.g., L262Q), and X274A(e.g., T274A), wherein each position corresponds to the position of thepolypeptide of SEQ ID NO: 2.

In another embodiment, the subtilase variants comprise X62D (e.g., N62D)and one or more substitutions selected from the group consisting ofX101H (e.g., S101H), X104T (e.g., V104T), X156D (e.g., S156D), X163G(e.g., S163G), X170S (e.g., R170S), X170L (e.g., R170L), X209W (e.g.,Y209W), X238E (e.g., N238E), and X262E (e.g., L262E); and optionally mayfurther comprise one or more alterations selected from the groupconsisting of X3T (e.g., S3T), X4I (e.g., V4I), X9C (e.g., S9C), X9D(e.g., S9D), X9E (e.g., S9E), X9Q (e.g., S9Q), X14T (e.g., A15T), X24G(e.g., S24G), X24R (e.g., S24R), X27R (e.g., K27R), *36D, X43A (e.g.,N43A), X43C (e.g., N43C), X43L (e.g., N43L), X43R (e.g., N43R), X43W(e.g., N43W), X68A (e.g., V68A), X72A (e.g., I72A), X72V (e.g., I72V),X76D (e.g., N76D), X78D (e.g., S78D), X87R (e.g., N87R), X87S (e.g.,N87S), *97E, X98S (e.g., A98S), X99A (e.g., S99A), X99D (e.g., S99D),X99A (e.g., S99A), X99D (e.g., S99D), X99E (e.g., S99E), X99G (e.g.,S99G), *99aD, X101D (e.g., S101D), X101E (e.g., S101E), X101G (e.g.,S101G), X101I (e.g., S101I), X101K (e.g., S101K), X101L (e.g., S101L),X101M (e.g., S101M), X101N (e.g., S101N), X101R (e.g., S101R), X103A(e.g., S103A), X104F (e.g., V104F), X104I (e.g., V104I), X104N (e.g.,V104N), X104Y (e.g., V104Y), X106A (e.g., S106A), X114V (e.g., A114V),X115T (e.g., G115T), X115W (e.g., G115W), X118R (e.g., G118R), X118V(e.g., G118V), X120D (e.g., H120D), X120I (e.g., H120I), X120N (e.g.,H120N), X120T (e.g., H120T), X120V (e.g., H120V), X123S (e.g., N123S),X128A (e.g., S128A), X128L (e.g., S128L), X128S (e.g., S128S), X129D(e.g., P129D), X129N (e.g., P129N), X129Q (e.g., P129Q), X130A (e.g.,S130A), X147W (e.g., V147W), X149C (e.g., V149C), X149N (e.g., V149N),X158E (e.g., A158E), X160D (e.g., G160D, X160P (e.g., G160P), X161C(e.g., S161C), X161E (e.g., S161E), X162L (e.g., I162L), X163A (e.g.,S163A), X163D (e.g., S163D), X167A (e.g., Y167A), X182C (e.g., Q182C),X182E (e.g., Q182E), X185C (e.g., N185C), X185E (e.g., N185E), X188C(e.g., S188C), X188D (e.g., S188D), X188E (e.g., S188E), X191N (e.g.,Q191N), X194P (e.g., A194P), X195E (e.g., G195E), X199M (e.g., V199M),X204D (e.g., N204D), X204V (e.g., N204V), X205I (e.g., V205I), X206C(e.g., Q206C), X206E (e.g., Q206E), X206I (e.g., Q206I), X206K (e.g.,Q206K), X206L (e.g., Q206L), X206T (e.g., Q206T), X206V (e.g., Q206V),X206W (e.g., Q206W), X209W (e.g., Y209W), X212A (e.g., S212A), X212D(e.g., S212D), X212G (e.g., S212G), X212N (e.g., S212N), X216I (e.g.,S216I), X216T (e.g., S216T), X216V (e.g., S216V), X217C (e.g., L217C),X217D (e.g., L217D), X217E (e.g., L217E), X217M (e.g., L217M), X217Q(e.g., L217Q), X217Y (e.g., L217Y), X218D (e.g., N218D), X218E (e.g.,N218E), X218T (e.g., N218T), X222C (e.g., M222C), X222R (e.g., M222R),X222S (e.g., M222S), X225A (e.g., P225A), X232V (e.g., A232V), X235L(e.g., K235L), X236H (e.g., Q236H), X245K (e.g., Q245K), X245R (e.g.,Q245R), X252K (e.g., N252K), X255C (e.g., T255C), X255E (e.g., T255E),X256A (e.g., S256A), X256C (e.g., S256C), X256D (e.g., S256D), X256V(e.g., S256V), X256Y (e.g., S256Y), X259D (e.g., S259D), X260E (e.g.,T260E), X260P (e.g., T260P), X261C (e.g., N261C), X261E (e.g., N261E),X261F (e.g., N261F), X261L (e.g., N261L), X261M (e.g., N261M), X261V(e.g., N261V), X261W (e.g., N261W), X261Y (e.g., N261Y), X262C (e.g.,L262C), X262E (e.g., L262E), X262Q (e.g., L262Q), and X274A (e.g.,T274A), wherein each position corresponds to the position of thepolypeptide of SEQ ID NO: 2. In another embodiment, the subtilasevariants comprise X62D+X245R+X248D (e.g., N62D+Q245R+N248D) and one ormore substitutions selected from the group consisting of X156D (e.g.,S156D), X163G (e.g., S163G), X163K (e.g., S163K), X170S (e.g., R170S),X209W (e.g., Y209W), and X262E (e.g., L262E); and optionally may furthercomprise one or more alterations selected from the group consisting ofX3T (e.g., S3T), X4I (e.g., V4I), X9C (e.g., S9C), X9D (e.g., S9D), X9E(e.g., S9E), X9Q (e.g., S9Q), X14T (e.g., A15T), X24G (e.g., S24G), X24R(e.g., S24R), X27R (e.g., K27R), *36D, X43A (e.g., N43A), X43C (e.g.,N43C), X43L (e.g., N43L), X43R (e.g., N43R), X43W (e.g., N43W), X68A(e.g., V68A), X72A (e.g., I72A), X72V (e.g., I72V), X76D (e.g., N76D),X78D (e.g., S78D), X87R (e.g., N87R), X87S (e.g., N87S), *97E, X98S(e.g., A98S), X99A (e.g., S99A), X99D (e.g., S99D), X99A (e.g., S99A),X99D (e.g., S99D), X99E (e.g., S99E), X99G (e.g., S99G),*99aD, X101D(e.g., S101D), X101E (e.g., S101E), X101G (e.g., S101G), X101I (e.g.,S101I), X101K (e.g., S101K), X101L (e.g., S101L), X101M (e.g., S101M),X101N (e.g., S101N), X101R (e.g., S101R), X103A (e.g., S103A), X104F(e.g., V104F), X104I (e.g., V104I), X104N (e.g., V104N), X104Y (e.g.,V104Y), X106A (e.g., S106A), X114V (e.g., A114V), X115T (e.g., G115T),X115W (e.g., G115W), X118R (e.g., G118R), X118V (e.g., G118V), X120D(e.g., H120D), X120I (e.g., H120I), X120N (e.g., H120N), X120T (e.g.,H120T), X120V (e.g., H120V), X123S (e.g., N123S), X128A (e.g., S128A),X128L (e.g., S128L), X128S (e.g., S128S), X129D (e.g., P129D), X129N(e.g., P129N), X129Q (e.g., P129Q), X130A (e.g., S130A), X147W (e.g.,V147W), X149C (e.g., V149C), X149N (e.g., V149N), X158E (e.g., A158E),X160D (e.g., G160D, X160P (e.g., G160P), X161C (e.g., S161C), X161E(e.g., S161E), X162L (e.g., I162L), X163A (e.g., S163A), X163D (e.g.,S163D), X167A (e.g., Y167A), X182C (e.g., Q182C), X182E (e.g., Q182E),X185C (e.g., N185C), X185E (e.g., N185E), X188C (e.g., S188C), X188D(e.g., S188D), X188E (e.g., S188E), X191N (e.g., Q191N), X194P (e.g.,A194P), X195E (e.g., G195E), X199M (e.g., V199M), X204D (e.g., N204D),X204V (e.g., N204V), X205I (e.g., V205I), X206C (e.g., Q206C), X206E(e.g., Q206E), X206I (e.g., Q206I), X206K (e.g., Q206K), X206L (e.g.,Q206L), X206T (e.g., Q206T), X206V (e.g., Q206V), X206W (e.g., Q206W),X209W (e.g., Y209W), X212A (e.g., S212A), X212D (e.g., S212D), X212G(e.g., S212G), X212N (e.g., S212N), X216I (e.g., S216I), X216T (e.g.,S216T), X216V (e.g., S216V), X217C (e.g., L217C), X217D (e.g., L217D),X217E (e.g., L217E), X217M (e.g., L217M), X217Q (e.g., L217Q), X217Y(e.g., L217Y), X218D (e.g., N218D), X218E (e.g., N218E), X218T (e.g.,N218T), X222C (e.g., M222C), X222R (e.g., M222R), X222S (e.g., M222S),X225A (e.g., P225A), X232V (e.g., A232V), X235L (e.g., K235L), X236H(e.g., Q236H), X252K (e.g., N252K), X255C (e.g., T255C), X255E (e.g.,T255E), X256A (e.g., S256A), X256C (e.g., S256C), X256D (e.g., S256D),X256V (e.g., S256V), X256Y (e.g., S256Y), X259D (e.g., S259D), X260E(e.g., T260E), X260P (e.g., T260P), X261C (e.g., N261C), X261E (e.g.,N261E), X261F (e.g., N261F), X261L (e.g., N261L), X261M (e.g., N261M),X261V (e.g., N261V), X261W (e.g., N261W), X261Y (e.g., N261Y), X262C(e.g., L262C), X262E (e.g., L262E), X262Q (e.g., L262Q), and X274A(e.g., T274A), wherein each position corresponds to the position of thepolypeptide of SEQ ID NO: 2.

In another embodiment, the subtilase variants comprise X170L, X170N,X170S (e.g., R170L, R170N, R170S) and one or more substitutions selectedfrom the group consisting of X57P (e.g., S57P), X167A (e.g., Y167A),X172E (e.g., A172E), X206E (e.g., Q206E); and optionally may furthercomprise one or more alterations selected from the group consisting ofX3T (e.g., S3T), X4I (e.g., V4I), X9C (e.g., S9C), X9D (e.g., S9D), X9E(e.g., S9E), X9Q (e.g., S9Q), X14T (e.g., A15T), X24G (e.g., S24G), X24R(e.g., S24R), X27R (e.g., K27R), *36D, X43A (e.g., N43A), X43C (e.g.,N43C), X43L (e.g., N43L), X43R (e.g., N43R), X43W (e.g., N43W), X68A(e.g., V68A), X72A (e.g., I72A), X72V (e.g., I72V), X76D (e.g., N76D),X78D (e.g., S78D), X87R (e.g., N87R), X87S (e.g., N87S), *97E, X98S(e.g., A98S), X99A (e.g., S99A), X99D (e.g., S99D), X99A (e.g., S99A),X99D (e.g., S99D), X99E (e.g., S99E), X99G (e.g., S99G), *99D, X101D(e.g., S101D), X101E (e.g., S101E), X101G (e.g., S101G), X101I (e.g.,S101I), X101K (e.g., S101K), X101L (e.g., S101L), X101M (e.g., S101M),X101N (e.g., S101N), X101R (e.g., S101R), X103A (e.g., S103A), X104F(e.g., V104F), X104I (e.g., V104I), X104N (e.g., V104N), X104Y (e.g.,V104Y), X106A (e.g., S106A), X114V (e.g., A114V), X115T (e.g., G115T),X115W (e.g., G115W), X118R (e.g., G118R), X118V (e.g., G118V), X120D(e.g., H120D), X120I (e.g., H120I), X120N (e.g., H120N), X120T (e.g.,H120T), X120V (e.g., H120V), X123S (e.g., N123S), X128A (e.g., S128A),X128L (e.g., S128L), X128S (e.g., S128S), X129D (e.g., P129D), X129N(e.g., P129N), X129Q (e.g., P129Q), X130A (e.g., S130A), X147W (e.g.,V147W), X149C (e.g., V149C), X149N (e.g., V149N), X158E (e.g., A158E),X160D (e.g., G160D, X160P (e.g., G160P), X161C (e.g., S161C), X161E(e.g., S161E), X162L (e.g., I162L), X163A (e.g., S163A), X163D (e.g.,S163D), X167A (e.g., Y167A), X182C (e.g., Q182C), X182E (e.g., Q182E),X185C (e.g., N185C), X185E (e.g., N185E), X188C (e.g., S188C), X188D(e.g., S188D), X188E (e.g., S188E), X191N (e.g., Q191N), X194P (e.g.,A194P), X195E (e.g., G195E), X199M (e.g., V199M), X204D (e.g., N204D),X204V (e.g., N204V), X205I (e.g., V205I), X206C (e.g., Q206C), X206E(e.g., Q206E), X206I (e.g., Q206I), X206K (e.g., Q206K), X206L (e.g.,Q206L), X206T (e.g., Q206T), X206V (e.g., Q206V), X206W (e.g., Q206W),X209W (e.g., Y209W), X212A (e.g., S212A), X212D (e.g., S212D), X212G(e.g., S212G), X212N (e.g., S212N), X216I (e.g., S216I), X216T (e.g.,S216T), X216V (e.g., S216V), X217C (e.g., L217C), X217D (e.g., L217D),X217E (e.g., L217E), X217M (e.g., L217M), X217Q (e.g., L217Q), X217Y(e.g., L217Y), X218D (e.g., N218D), X218E (e.g., N218E), X218T (e.g.,N218T), X222C (e.g., M222C), X222R (e.g., M222R), X222S (e.g., M222S),X225A (e.g., P225A), X232V (e.g., A232V), X235L (e.g., K235L), X236H(e.g., Q236H), X252K (e.g., N252K), X255C (e.g., T255C), X255E (e.g.,T255E), X256A (e.g., S256A), X256C (e.g., S256C), X256D (e.g., S256D),X256V (e.g., S256V), X256Y (e.g., S256Y), X259D (e.g., S259D), X260E(e.g., T260E), X260P (e.g., T260P), X261C (e.g., N261C), X261E (e.g.,N261E), X261F (e.g., N261F), X261L (e.g., N261L), X261M (e.g., N261M),X261V (e.g., N261V), X261W (e.g., N261W), X261Y (e.g., N261Y), X262C(e.g., L262C), X262E (e.g., L262E), X262Q (e.g., L262Q), and X274A(e.g., T274A), wherein each position corresponds to the position of thepolypeptide of SEQ ID NO: 2.

In another embodiment, the subtilase variants comprise X99D (e.g., S99D)and one or more substitutions selected from the group consisting of*97aN, *98aA, X98T (e.g., A98T), X261D (e.g., N261D), and X262Q (e.g.,L262Q); and optionally may further comprise one or more alterationsselected from the group consisting of X3T (e.g., S3T), X4I (e.g., V4I),X9C (e.g., S9C), X9D (e.g., S9D), X9E (e.g., S9E), X9Q (e.g., S9Q), X14T(e.g., A15T), X24G (e.g., S24G), X24R (e.g., S24R), X27R (e.g., K27R),*36D, X43A (e.g., N43A), X43C (e.g., N43C), X43L (e.g., N43L), X43R(e.g., N43R), X43W (e.g., N43W), X68A (e.g., V68A), X72A (e.g., I72A),X72V (e.g., I72V), X76D (e.g., N76D), X78D (e.g., S78D), X87R (e.g.,N87R), X87S (e.g., N87S), *97E, X98S (e.g., A98S), X99A (e.g., S99A),X99D (e.g., S99D), X99A (e.g., S99A), X99D (e.g., S99D), X99E (e.g.,S99E), X99G (e.g., S99G), *99D, X101D (e.g., S101D), X101E (e.g.,S101E), X101G (e.g., S101G), X101I (e.g., S101I), X101K (e.g., S101K),X101L (e.g., S101L), X101M (e.g., S101M), X101N (e.g., S101N), X101R(e.g., S101R), X103A (e.g., S103A), X104F (e.g., V104F), X104I (e.g.,V104I), X104N (e.g., V104N), X104Y (e.g., V104Y), X106A (e.g., S106A),X114V (e.g., A114V), X115T (e.g., G115T), X115W (e.g., G115W), X118R(e.g., G118R), X118V (e.g., G118V), X120D (e.g., H120D), X120I (e.g.,H120I), X120N (e.g., H120N), X120T (e.g., H120T), X120V (e.g., H120V),X123S (e.g., N123S), X128A (e.g., S128A), X128L (e.g., S128L), X128S(e.g., S128S), X129D (e.g., P129D), X129N (e.g., P129N), X129Q (e.g.,P129Q), X130A (e.g., S130A), X147W (e.g., V147W), X149C (e.g., V149C),X149N (e.g., V149N), X158E (e.g., A158E), X160D (e.g., G160D, X160P(e.g., G160P), X161C (e.g., S161C), X161E (e.g., S161E), X162L (e.g.,I162L), X163A (e.g., S163A), X163D (e.g., S163D), X167A (e.g., Y167A),X182C (e.g., Q182C), X182E (e.g., Q182E), X185C (e.g., N185C), X185E(e.g., N185E), X188C (e.g., S188C), X188D (e.g., S188D), X188E (e.g.,S188E), X191N (e.g., Q191N), X194P (e.g., A194P), X195E (e.g., G195E),X199M (e.g., V199M), X204D (e.g., N204D), X204V (e.g., N204V), X205I(e.g., V205I), X206C (e.g., Q206C), X206E (e.g., Q206E), X206I (e.g.,Q206I), X206K (e.g., Q206K), X206L (e.g., Q206L), X206T (e.g., Q206T),X206V (e.g., Q206V), X206W (e.g., Q206W), X209W (e.g., Y209W), X212A(e.g., S212A), X212D (e.g., S212D), X212G (e.g., S212G), X212N (e.g.,S212N), X216I (e.g., S216I), X216T (e.g., S216T), X216V (e.g., S216V),X217C (e.g., L217C), X217D (e.g., L217D), X217E (e.g., L217E), X217M(e.g., L217M), X217Q (e.g., L217Q), X217Y (e.g., L217Y), X218D (e.g.,N218D), X218E (e.g., N218E), X218T (e.g., N218T), X222C (e.g., M222C),X222R (e.g., M222R), X222S (e.g., M222S), X225A (e.g., P225A), X232V(e.g., A232V), X235L (e.g., K235L), X236H (e.g., Q236H), X252K (e.g.,N252K), X255C (e.g., T255C), X255E (e.g., T255E), X256A (e.g., S256A),X256C (e.g., S256C), X256D (e.g., S256D), X256V (e.g., S256V), X256Y(e.g., S256Y), X259D (e.g., S259D), X260E (e.g., T260E), X260P (e.g.,T260P), X261C (e.g., N261C), X261E (e.g., N261E), X261F (e.g., N261F),X261L (e.g., N261L), X261M (e.g., N261M), X261V (e.g., N261V), X261W(e.g., N261W), X261Y (e.g., N261Y), X262C (e.g., L262C), X262E (e.g.,L262E), X262Q (e.g., L262Q), and X274A (e.g., T274A), wherein eachposition corresponds to the position of the polypeptide of SEQ ID NO: 2.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and one or moresubstitutions selected from the group consisting of X59D (e.g., Q59D),X62D (e.g., N62D), X76D (e.g., N76D), X104T (e.g., V104T), X120D (e.g.,H120D), X133P (e.g. A133P), X141N (e.g. S141N), X156D (e.g., S156D),X163G (e.g., S163G), X209W (e.g., Y209W), X228V (e.g. A228V), X230V(e.g., A230V), X238E (e.g., N238E), X261D (e.g., N261D), and X262E(e.g., L262E), wherein

(i) the positions correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X59D (e.g., Q59D), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X62D (e.g., N62D), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X76D (e.g., N76D), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X104T (e.g., V104T), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X120D (e.g., H120D), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X133P (e.g., A133P, wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X141N (e.g., S141N), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X156D (e.g., S156D), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X163G (e.g., S163G), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X209W (e.g., Y209W), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X228V (e.g., A228V), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X230V (e.g., A230V), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X238E (e.g., N238E), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X261D (e.g., N261D), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and thesubstitution X262E (e.g., L262E), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and one or more substitutions selectedfrom the group consisting of X21D (e.g., L21D), X59D (e.g., Q59D), X101H(e.g., S101H), X120D (e.g., H120D), X156D (e.g., S156D), X163G (e.g.,S163G), X194P (e.g., A194P), X195E (e.g., G195E), X209W (e.g., Y209W),X238E (e.g., N238E), X256D (e.g. N256D), X261D (e.g., N261D), and X262E(e.g., L262E), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X21D (e.g., L21D),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X59D (e.g., Q59D),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X101H (e.g., S101H),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X120D (e.g., H120D),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X156D (e.g., S156D),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X163G (e.g., S163G),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X194P (e.g., A194P),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X195E (e.g., G195E),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X209W (e.g., Y209W),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X238E (e.g., N238E),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X256D (e.g. N256D),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X261D (e.g., N261D),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the insertion *99aE and the substitution X262E (e.g., L262E),wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X62D (e.g., N62D) and one or moresubstitutions selected from the group consisting of X101H (e.g., S101H),X104T (e.g., V104T), X156D (e.g., S156D), X163G (e.g., S163G), X170S,X170L (e.g., R170S, R170L), X209W (e.g., Y209W), X238E (e.g., N238E),X245R (e.g. Q245R) and X262E (e.g., L262E), wherein

(i) the positions correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X62D (e.g., N62D) and the substitution X101H(e.g., S101H), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X62D (e.g., N62D) and the substitution X104T(e.g., V104T), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X62D (e.g., N62D) and the substitution X156D(e.g., S156D), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X62D (e.g., N62D) and the substitution X163G(e.g., S163G), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X62D (e.g., N62D) and the substitution X170S,or X170L (e.g., R170S or R170L), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X62D (e.g., N62D) and the substitution X209W(e.g., Y209W), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X62D (e.g., N62D) and the substitution X238E(e.g., N238E), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X62D (e.g., N62D) and the substitution X245R(e.g. Q245R) wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X62D (e.g., N62D) and the substitution X262E(e.g., L262E), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X62D+X245R+X248D (e.g., N62D+Q245R+N248D)and one or more substitutions selected from the group consisting ofX156D (e.g., S156D), X163G (e.g., S163G), X163K (e.g., S163K), X170S(e.g., R170S), X209W (e.g., Y209W), and X262E (e.g., L262E), wherein (i)the positions correspond to the positions of the polypeptide of SEQ IDNO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X62D+X245R+X248D (e.g., N62D+Q245R+N248D)and the substitution X156D (e.g., S156D), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X62D+X245R+X248D (e.g., N62D+Q245R+N248D)and the substitution X163G (e.g., S163G), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X62D+X245R+X248D (e.g., N62D+Q245R+N248D)and the substitution X163K (e.g., S163K), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X62D+X245R+X248D (e.g., N62D+Q245R+N248D)and the substitution X170S (e.g., R170S), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X62D+X245R+X248D (e.g., N62D+Q245R+N248D)and the substitution X209W (e.g., Y209W), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X62D+X245R+X248D (e.g., N62D+Q245R+N248D)and the substitution X262E (e.g., L262E), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X170L, X170N or X170S (e.g. R170L, R170N,R170S) and one or more substitutions selected from the group consistingof X57P (e.g. S57P), X167A (e.g. Y167A), X172E (e.g. A172E), X206E (e.g.Q206E), wherein

(i) the positions correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X170L, X170N or X170S (e.g. R170L, R170N,R170S) and the substitution X57P (e.g. S57P), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X170L, X170N or X170S (e.g. R170L, R170N,R170S) and the substitution X167A (e.g. Y167A), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X170L, X170N or X170S (e.g. R170L, R170N,R170S) and the substitution, X172E (e.g. A172E), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitutions X170L, X170N or X170S (e.g. R170L, R170N,R170S) and the substitution X206E (e.g. Q206E), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X99D (e.g. S99D) and one or more alterationsselected from the group consisting of *97aN, *98aA, X98T (e.g. A98T),X261D (e.g., N261D), and X262Q (e.g., L262Q), wherein (i) the positionscorrespond to the positions of the polypeptide of SEQ ID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X99D (e.g. S99D) and the insertion *97aN,wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X99D (e.g. S99D) and the insertion *98aA,wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X99D (e.g. S99D) and the substitution X98T(e.g. A98T), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X99D (e.g. S99D) and the substitution X261D(e.g., N261D), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity; and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

One embodiment of the invention relates to a subtilase variant, whichcomprises the substitution X99D (e.g. S99D) and the substitution X262Q(e.g., L262Q), wherein

(i) the position correspond to the positions of the polypeptide of SEQID NO: 2;

(ii) the variant has protease activity and

(iii) the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11 and wherein optionally the variant has an improved washperformance compared to SEQ ID NO: 1 when measured in AMSA assay.

The present invention relates to subtilase variants having proteaseactivity and comprising

(a) X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and one or moresubstitutions selected from the group consisting of X59D (e.g., Q59D),X62D (e.g., N62D), X76D (e.g., N76D), X104T (e.g., V104T), X120D (e.g.,H120D), X133P (e.g., A133P), X141N (e.g., S141N), X156D (e.g., S156D),X163G (e.g., S163G), X209W (e.g., Y209W), X228V (e.g., A228V), X230V(e.g., A230V), X238E (e.g., N238E), X261D (e.g., N261D), and X262E(e.g., L262E);

(b)*99aE and one or more substitutions selected from the groupconsisting of X21D (e.g. L21D), X59D (e.g., Q59D), X101H (e.g., S101H),X120D (e.g., H120D), X156D (e.g., S156D), X163G (e.g., S163G), X194P(e.g., A194P), X195E (e.g., G195E), X209W (e.g., Y209W), X238E (e.g.,N238E), X256D (e.g., N256D), X261D (e.g., N261D), and X262E (e.g.,L262E);

(c) X62D (e.g., N62D) and one or more substitutions selected from thegroup consisting of X101H (e.g., S101H), X104T (e.g., V104T), X156D(e.g., S156D), X163G (e.g., S163G), X170S (e.g., R170S), X170L (e.g.,R170L), X209W (e.g., Y209W), X238E (e.g., N238E), X245R (e.g. Q245R) andX262E (e.g., L262E);

(d) X62D+X245R+X248D (e.g., N62D+Q245R+N248D) and one or moresubstitutions selected from the group consisting of X156D (e.g., S156D),X163G (e.g., S163G), X163K (e.g., S163K), X170S (e.g., R170S), X209W(e.g., Y209W), and X262E (e.g., L262E);

(e) X170L, X170N or X170S (e.g. R170L, R170N or R170S) and one or moresubstitutions selected from the group consisting of X57P (e.g. S57P),X167A (e.g. Y167A), X172E (e.g. A172E), X206E (e.g. Q206E),

(f) X99D (e.g. S99D) and one or more substitutions selected from thegroup consisting of *97aN, *98aA, X98T (e.g., A98T), X261D (e.g.,N261D), and X262Q (e.g., L262Q), wherein the positions correspond to thepositions of the polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant has improved stability, inparticular improved storage stability, compared to the parent subtilase.In a preferred embodiment, the subtilase variant has improved stability,in particular improved storage stability, and on par or improved washperformance compared to the parent subtilase.

In another embodiment, the subtilase variant is

a) a polypeptide that has at least 60% but less than 100% sequenceidentity to the amino acid sequence of the parent subtilase;

b) a polypeptide that is encoded by a polynucleotide that hybridizesunder low stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with:

-   -   (i) the mature polypeptide coding sequence of the parent        subtilase or    -   (ii) the full-length complement of (i); or

c) a polypeptide that is encoded by a polynucleotide having at least 60%but less than 100% sequence identity to the mature polypeptide codingsequence of the parent subtilase.

In an embodiment, the subtilase variant has at least 65% but less than100% sequence identity to the parent subtilase. In an embodiment, thesubtilase variant has at least 70% but less than 100% sequence identityto the parent subtilase. In an embodiment, the subtilase variant has atleast 75% but less than 100% sequence identity to the parent subtilase.In an embodiment, the subtilase variant has at least 80% but less than100% sequence identity to the parent subtilase. In an embodiment, thesubtilase variant has at least 85% but less than 100% sequence identityto the parent subtilase. In an embodiment, the subtilase variant has atleast 90% but less than 100% sequence identity to the parent subtilase.In an embodiment, the subtilase variant has at least 93% but less than100% sequence identity to the parent subtilase. In an embodiment, thesubtilase variant has at least 95% but less than 100% sequence identityto the parent subtilase. In an embodiment, the subtilase variant has atleast 96% but less than 100% sequence identity to the parent subtilase.In an embodiment, the subtilase variant has at least 97% but less than100% sequence identity to the parent subtilase. In an embodiment, thesubtilase variant has at least 98% but less than 100% sequence identityto the parent subtilase.

In an embodiment, the variant has an amino acid sequence which is atleast 60% identical to SEQ ID NO: 1, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has an amino acid sequence which isat least 60% identical to SEQ ID NO: 2, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has an amino acid sequence which isat least 60% identical to SEQ ID NO: 3, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 3. Inanother embodiment, the variant has an amino acid sequence which is atleast 60% identical to SEQ ID NO: 4, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 4.

In another embodiment, the variant has an amino acid sequence which isat least 60% identical to SEQ ID NO: 5, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 5.

In another embodiment, the variant has an amino acid sequence which isat least 60% identical to SEQ ID NO: 6, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 6.

In another embodiment, the variant has an amino acid sequence which isat least 60% identical to SEQ ID NO: 7, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 7.

In another embodiment, the variant has an amino acid sequence which isat least 60% identical to SEQ ID NO: 8, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 8. Inanother embodiment, the variant has an amino acid sequence which is atleast 60% identical to SEQ ID NO: 9, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 9.

In another embodiment, the variant has an amino acid sequence which isat least 60% identical to SEQ ID NO: 10, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 10.

In another embodiment, the variant has an amino acid sequence which isat least 60% identical to SEQ ID NO: 11, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 11.

In one aspect, the total number of alterations in the parent subtilaseis between 3 and 30, preferably between 3 and 20, more preferablybetween 3 and 15, even more preferably between 3 and 10, most preferablybetween 3 and 8 alterations. In another aspect, total number ofalterations in the parent subtilase is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30alterations.

The subtilase variants of the present invention may further one or moreadditional alterations. The amino acid changes may be of a minor nature,that is conservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for protease activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. For BPN′ (SEQ ID NO: 2) the catalytictriad comprising the amino acids S221, H64, and D32 is essential forprotease activity of the enzyme.

In an embodiment, the subtilase variants of the present inventioncomprise X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and one or moresubstitutions selected from the group consisting of X59D (e.g., Q59D),X76D (e.g., N76D), X104T (e.g., V104T), X120D (e.g., H120D), X156D(e.g., S156D), X163G (e.g., S163G), X209W (e.g., Y209W), X238E (e.g.,N238E), X261D (e.g., N261D), and X262E (e.g., L262E); and optionally mayfurther comprise one or alterations selected from the group consistingof X3T (e.g., S3T), X4I (e.g., V4I), X9C (e.g., S9C), X9D (e.g., S9D),X9E (e.g., S9E), X9Q (e.g., S9Q), X14T (e.g., A15T), X24G (e.g., S24G),X24R (e.g., S24R), X27R (e.g., K27R), *36D, X43A (e.g., N43A), X43C(e.g., N43C), X43L (e.g., N43L), X43R (e.g., N43R), X43W (e.g., N43W),X68A (e.g., V68A), X72A (e.g., I72A), X72V (e.g., I72V), X76D (e.g.,N76D), X78D (e.g., S78D), X87R (e.g., N87R), X87S (e.g., N87S), *97E,X98S (e.g., A98S), X99A (e.g., S99A), X99D (e.g., S99D), X99A (e.g.,S99A), X99D (e.g., S99D), X99E (e.g., S99E), X99G (e.g., S99G), *99D,X101D (e.g., S101D), X101E (e.g., S101E), X101G (e.g., S101G), X101I(e.g., S101I), X101K (e.g., S101K), X101L (e.g., S101L), X101M (e.g.,S101M), X101N (e.g., S101N), X101R (e.g., S101R), X103A (e.g., S103A),X104F (e.g., V104F), X104I (e.g., V104I), X104N (e.g., V104N), X104Y(e.g., V104Y), X106A (e.g., S106A), X114V (e.g., A114V), X115T (e.g.,G115T), X115W (e.g., G115W), X118R (e.g., G118R), X118V (e.g., G118V),X120D (e.g., H120D), X120I (e.g., H120I), X120N (e.g., H120N), X120T(e.g., H120T), X120V (e.g., H120V), X123S (e.g., N123S), X128A (e.g.,S128A), X128L (e.g., S128L), X128S (e.g., S128S), X129D (e.g., P129D),X129N (e.g., P129N), X129Q (e.g., P129Q), X130A (e.g., S130A), X147W(e.g., V147W), X149C (e.g., V149C), X149N (e.g., V149N), X158E (e.g.,A158E), X160D (e.g., G160D, X160P (e.g., G160P), X161C (e.g., S161C),X161E (e.g., S161E), X162L (e.g., I162L), X163A (e.g., S163A), X163D(e.g., S163D), X182C (e.g., Q182C), X182E (e.g., Q182E), X185C (e.g.,N185C), X185E (e.g., N185E), X188C (e.g., S188C), X188D (e.g., S188D),X188E (e.g., S188E), X191N (e.g., Q191N), X195E (e.g., G195E), X199M(e.g., V199M), X204D (e.g., N204D), X204V (e.g., N204V), X205I (e.g.,V205I), X206C (e.g., Q206C), X206E (e.g., Q206E), X206I (e.g., Q206I),X206K (e.g., Q206K), X206L (e.g., Q206L), X206T (e.g., Q206T), X206V(e.g., Q206V), X206W (e.g., Q206W), X209W (e.g., Y209W), X212A (e.g.,S212A), X212D (e.g., S212D), X212G (e.g., S212G), X212N (e.g., S212N),X216I (e.g., S216I), X216T (e.g., S216T), X216V (e.g., S216V), X217C(e.g., L217C), X217D (e.g., L217D), X217E (e.g., L217E), X217M (e.g.,L217M), X217Q (e.g., L217Q), X217Y (e.g., L217Y), X218D (e.g., N218D),X218E (e.g., N218E), X218T (e.g., N218T), X222C (e.g., M222C), X222R(e.g., M222R), X222S (e.g., M222S), X225A (e.g., P225A), X232V (e.g.,A232V), X235L (e.g., K235L), X236H (e.g., Q236H), X245K (e.g., Q245K),X245R (e.g., Q245R), X252K (e.g., N252K), X255C (e.g., T255C), X255E(e.g., T255E), X256A (e.g., S256A), X256C (e.g., S256C), X256D (e.g.,S256D), X256V (e.g., S256V), X256Y (e.g., S256Y), X259D (e.g., S259D),X260E (e.g., T260E), X260P (e.g., T260P), X261C (e.g., N261C), X261E(e.g., N261E), X261F (e.g., N261F), X261L (e.g., N261L), X261M (e.g.,N261M), X261V (e.g., N261V), X261W (e.g., N261W), X261Y (e.g., N261Y),X262C (e.g., L262C), X262E (e.g., L262E), X262Q (e.g., L262Q), and X274A(e.g., T274A), wherein each position corresponds to the position of thepolypeptide of SEQ ID NO: 2.

In another embodiment, the subtilase variants of the present inventioncomprise *99aE and one or more substitutions selected from the groupconsisting of X59D (e.g., Q59D), X101H (e.g., S101H), X120D (e.g.,H120D), X156D (e.g., S156D), X163G (e.g., S163G), X194P (e.g., A194P),X195E (e.g., G195E), X209W (e.g., Y209W), X238E (e.g., N238E), X261D(e.g., N261D), and X262E (e.g., L262E); and optionally may furthercomprise one or more alterations selected from the group consisting ofX3T (e.g., S3T), X4I (e.g., V4I), X9C (e.g., S9C), X9D (e.g., S9D), X9E(e.g., S9E), X9Q (e.g., S9Q), X14T (e.g., A15T), X24G (e.g., S24G), X24R(e.g., S24R), X27R (e.g., K27R), *36D, X43A (e.g., N43A), X43C (e.g.,N43C), X43L (e.g., N43L), X43R (e.g., N43R), X43W (e.g., N43W), X68A(e.g., V68A), X72A (e.g., I72A), X72V (e.g., I72V), X76D (e.g., N76D),X78D (e.g., S78D), X87R (e.g., N87R), X87S (e.g., N87S), *97E, X98S(e.g., A98S), X99A (e.g., S99A), X99D (e.g., S99D), X99A (e.g., S99A),X99D (e.g., S99D), X99E (e.g., S99E), X99G (e.g., S99G), X101D (e.g.,S101D), X101E (e.g., S101E), X101G (e.g., S101G), X101I (e.g., S101I),X101K (e.g., S101K), X101L (e.g., S101L), X101M (e.g., S101M), X101N(e.g., S101N), X101R (e.g., S101R), X103A (e.g., S103A), X104F (e.g.,V104F), X104I (e.g., V104I), X104N (e.g., V104N), X104Y (e.g., V104Y),X106A (e.g., S106A), X114V (e.g., A114V), X115T (e.g., G115T), X115W(e.g., G115W), X118R (e.g., G118R), X118V (e.g., G118V), X120D (e.g.,H120D), X120I (e.g., H120I), X120N (e.g., H120N), X120T (e.g., H120T),X120V (e.g., H120V), X123S (e.g., N123S), X128A (e.g., S128A), X128L(e.g., S128L), X128S (e.g., S128S), X129D (e.g., P129D), X129N (e.g.,P129N), X129Q (e.g., P129Q), X130A (e.g., S130A), X147W (e.g., V147W),X149C (e.g., V149C), X149N (e.g., V149N), X158E (e.g., A158E), X160D(e.g., G160D, X160P (e.g., G160P), X161C (e.g., S161C), X161E (e.g.,S161E), X162L (e.g., I162L), X163A (e.g., S163A), X163D (e.g., S163D),X167A (e.g., Y167A), X170S (e.g., R170S), X182C (e.g., Q182C), X182E(e.g., Q182E), X185C (e.g., N185C), X185E (e.g., N185E), X188C (e.g.,S188C), X188D (e.g., S188D), X188E (e.g., S188E), X191N (e.g., Q191N),X194P (e.g., A194P), X195E (e.g., G195E), X199M (e.g., V199M), X204D(e.g., N204D), X204V (e.g., N204V), X205I (e.g., V205I), X206C (e.g.,Q206C), X206E (e.g., Q206E), X206I (e.g., Q206I), X206K (e.g., Q206K),X206L (e.g., Q206L), X206T (e.g., Q206T), X206V (e.g., Q206V), X206W(e.g., Q206W), X209W (e.g., Y209W), X212A (e.g., S212A), X212D (e.g.,S212D), X212G (e.g., S212G), X212N (e.g., S212N), X216I (e.g., S216I),X216T (e.g., S216T), X216V (e.g., S216V), X217C (e.g., L217C), X217D(e.g., L217D), X217E (e.g., L217E), X217M (e.g., L217M), X217Q (e.g.,L217Q), X217Y (e.g., L217Y), X218D (e.g., N218D), X218E (e.g., N218E),X218T (e.g., N218T), X222C (e.g., M222C), X222R (e.g., M222R), X222S(e.g., M222S), X225A (e.g., P225A), X232V (e.g., A232V), X235L (e.g.,K235L), X236H (e.g., Q236H), X245K (e.g., Q245K), X245R (e.g., Q245R),X252K (e.g., N252K), X255C (e.g., T255C), X255E (e.g., T255E), X256A(e.g., S256A), X256C (e.g., S256C), X256D (e.g., S256D), X256V (e.g.,S256V), X256Y (e.g., S256Y), X259D (e.g., S259D), X260E (e.g., T260E),X260P (e.g., T260P), X261C (e.g., N261C), X261E (e.g., N261E), X261F(e.g., N261F), X261L (e.g., N261L), X261M (e.g., N261M), X261V (e.g.,N261V), X261W (e.g., N261W), X261Y (e.g., N261Y), X262C (e.g., L262C),X262E (e.g., L262E), X262Q (e.g., L262Q), and X274A (e.g., T274A),wherein each position corresponds to the position of the polypeptide ofSEQ ID NO: 2.

In another embodiment, the subtilase variants of the present inventioncomprise X62D (e.g., N62D) and one or more substitutions selected fromthe group consisting of X101H (e.g., S101H), X104T (e.g., V104T), X156D(e.g., S156D), X163G (e.g., S163G), X170S (e.g., R170S), X209W (e.g.,Y209W), X238E (e.g., N238E), and X262E (e.g., L262E); and optionally mayfurther comprise one or more alterations selected from the groupconsisting of X3T (e.g., S3T), X4I (e.g., V4I), X9C (e.g., S9C), X9D(e.g., S9D), X9E (e.g., S9E), X9Q (e.g., S9Q), X14T (e.g., A15T), X24G(e.g., S24G), X24R (e.g., S24R), X27R (e.g., K27R), *36D, X43A (e.g.,N43A), X43C (e.g., N43C), X43L (e.g., N43L), X43R (e.g., N43R), X43W(e.g., N43W), X68A (e.g., V68A), X72A (e.g., I72A), X72V (e.g., I72V),X76D (e.g., N76D), X78D (e.g., S78D), X87R (e.g., N87R), X87S (e.g.,N87S), *97E, X98S (e.g., A98S), X99A (e.g., S99A), X99D (e.g., S99D),X99A (e.g., S99A), X99D (e.g., S99D), X99E (e.g., S99E), X99G (e.g.,S99G), *99D, X101D (e.g., S101D), X101E (e.g., S101E), X101G (e.g.,S101G), X101I (e.g., S101I), X101K (e.g., S101K), X101L (e.g., S101L),X101M (e.g., S101M), X101N (e.g., S101N), X101R (e.g., S101R), X103A(e.g., S103A), X104F (e.g., V104F), X104I (e.g., V104I), X104N (e.g.,V104N), X104Y (e.g., V104Y), X106A (e.g., S106A), X114V (e.g., A114V),X115T (e.g., G115T), X115W (e.g., G115W), X118R (e.g., G118R), X118V(e.g., G118V), X120D (e.g., H120D), X120I (e.g., H120I), X120N (e.g.,H120N), X120T (e.g., H120T), X120V (e.g., H120V), X123S (e.g., N123S),X128A (e.g., S128A), X128L (e.g., S128L), X128S (e.g., S128S), X129D(e.g., P129D), X129N (e.g., P129N), X129Q (e.g., P129Q), X130A (e.g.,S130A), X147W (e.g., V147W), X149C (e.g., V149C), X149N (e.g., V149N),X158E (e.g., A158E), X160D (e.g., G160D, X160P (e.g., G160P), X161C(e.g., S161C), X161E (e.g., S161E), X162L (e.g., I162L), X163A (e.g.,S163A), X163D (e.g., S163D), X167A (e.g., Y167A), X182C (e.g., Q182C),X182E (e.g., Q182E), X185C (e.g., N185C), X185E (e.g., N185E), X188C(e.g., S188C), X188D (e.g., S188D), X188E (e.g., S188E), X191N (e.g.,Q191N), X194P (e.g., A194P), X195E (e.g., G195E), X199M (e.g., V199M),X204D (e.g., N204D), X204V (e.g., N204V), X205I (e.g., V205I), X206C(e.g., Q206C), X206E (e.g., Q206E), X206I (e.g., Q206I), X206K (e.g.,Q206K), X206L (e.g., Q206L), X206T (e.g., Q206T), X206V (e.g., Q206V),X206W (e.g., Q206W), X209W (e.g., Y209W), X212A (e.g., S212A), X212D(e.g., S212D), X212G (e.g., S212G), X212N (e.g., S212N), X216I (e.g.,S216I), X216T (e.g., S216T), X216V (e.g., S216V), X217C (e.g., L217C),X217D (e.g., L217D), X217E (e.g., L217E), X217M (e.g., L217M), X217Q(e.g., L217Q), X217Y (e.g., L217Y), X218D (e.g., N218D), X218E (e.g.,N218E), X218T (e.g., N218T), X222C (e.g., M222C), X222R (e.g., M222R),X222S (e.g., M222S), X225A (e.g., P225A), X232V (e.g., A232V), X235L(e.g., K235L), X236H (e.g., Q236H), X245K (e.g., Q245K), X245R (e.g.,Q245R), X252K (e.g., N252K), X255C (e.g., T255C), X255E (e.g., T255E),X256A (e.g., S256A), X256C (e.g., S256C), X256D (e.g., S256D), X256V(e.g., S256V), X256Y (e.g., S256Y), X259D (e.g., S259D), X260E (e.g.,T260E), X260P (e.g., T260P), X261C (e.g., N261C), X261E (e.g., N261E),X261F (e.g., N261F), X261L (e.g., N261L), X261M (e.g., N261M), X261V(e.g., N261V), X261W (e.g., N261W), X261Y (e.g., N261Y), X262C (e.g.,L262C), X262E (e.g., L262E), X262Q (e.g., L262Q), and X274A (e.g.,T274A), wherein each position corresponds to the position of thepolypeptide of SEQ ID NO: 2.

In another embodiment, the subtilase variants of the present inventioncomprise X62D+X245R+X248D (e.g., N62D+Q245R+N248D) and one or moresubstitutions selected from the group consisting of X156D (e.g., S156D),X163G (e.g., S163G), X163K (e.g., S163K), X170S (e.g., R170S), X209W(e.g., Y209W), and X262E (e.g., L262E); and optionally may furthercomprise one or more alterations selected from the group consisting ofX3T (e.g., S3T), X4I (e.g., V4I), X9C (e.g., S9C), X9D (e.g., S9D), X9E(e.g., S9E), X9Q (e.g., S9Q), X14T (e.g., A15T), X24G (e.g., S24G), X24R(e.g., S24R), X27R (e.g., K27R), *36D, X43A (e.g., N43A), X43C (e.g.,N43C), X43L (e.g., N43L), X43R (e.g., N43R), X43W (e.g., N43W), X68A(e.g., V68A), X72A (e.g., I72A), X72V (e.g., I72V), X76D (e.g., N76D),X78D (e.g., S78D), X87R (e.g., N87R), X87S (e.g., N87S), *97E, X98S(e.g., A98S), X99A (e.g., S99A), X99D (e.g., S99D), X99A (e.g., S99A),X99D (e.g., S99D), X99E (e.g., S99E), X99G (e.g., S99G), *99D, X101D(e.g., S101D), X101E (e.g., S101E), X101G (e.g., S101G), X101I (e.g.,S101I), X101K (e.g., S101K), X101L (e.g., S101L), X101M (e.g., S101M),X101N (e.g., S101N), X101R (e.g., S101R), X103A (e.g., S103A), X104F(e.g., V104F), X104I (e.g., V104I), X104N (e.g., V104N), X104Y (e.g.,V104Y), X106A (e.g., S106A), X114V (e.g., A114V), X115T (e.g., G115T),X115W (e.g., G115W), X118R (e.g., G118R), X118V (e.g., G118V), X120D(e.g., H120D), X120I (e.g., H120I), X120N (e.g., H120N), X120T (e.g.,H120T), X120V (e.g., H120V), X123S (e.g., N123S), X128A (e.g., S128A),X128L (e.g., S128L), X128S (e.g., S128S), X129D (e.g., P129D), X129N(e.g., P129N), X129Q (e.g., P129Q), X130A (e.g., S130A), X147W (e.g.,V147W), X149C (e.g., V149C), X149N (e.g., V149N), X158E (e.g., A158E),X160D (e.g., G160D, X160P (e.g., G160P), X161C (e.g., S161C), X161E(e.g., S161E), X162L (e.g., I162L), X163A (e.g., S163A), X163D (e.g.,S163D), X167A (e.g., Y167A), X182C (e.g., Q182C), X182E (e.g., Q182E),X185C (e.g., N185C), X185E (e.g., N185E), X188C (e.g., S188C), X188D(e.g., S188D), X188E (e.g., S188E), X191N (e.g., Q191N), X194P (e.g.,A194P), X195E (e.g., G195E), X199M (e.g., V199M), X204D (e.g., N204D),X204V (e.g., N204V), X205I (e.g., V205I), X206C (e.g., Q206C), X206E(e.g., Q206E), X206I (e.g., Q206I), X206K (e.g., Q206K), X206L (e.g.,Q206L), X206T (e.g., Q206T), X206V (e.g., Q206V), X206W (e.g., Q206W),X209W (e.g., Y209W), X212A (e.g., S212A), X212D (e.g., S212D), X212G(e.g., S212G), X212N (e.g., S212N), X216I (e.g., S216I), X216T (e.g.,S216T), X216V (e.g., S216V), X217C (e.g., L217C), X217D (e.g., L217D),X217E (e.g., L217E), X217M (e.g., L217M), X217Q (e.g., L217Q), X217Y(e.g., L217Y), X218D (e.g., N218D), X218E (e.g., N218E), X218T (e.g.,N218T), X222C (e.g., M222C), X222R (e.g., M222R), X222S (e.g., M222S),X225A (e.g., P225A), X232V (e.g., A232V), X235L (e.g., K235L), X236H(e.g., Q236H), X252K (e.g., N252K), X255C (e.g., T255C), X255E (e.g.,T255E), X256A (e.g., S256A), X256C (e.g., S256C), X256D (e.g., S256D),X256V (e.g., S256V), X256Y (e.g., S256Y), X259D (e.g., S259D), X260E(e.g., T260E), X260P (e.g., T260P), X261C (e.g., N261C), X261E (e.g.,N261E), X261F (e.g., N261F), X261L (e.g., N261L), X261M (e.g., N261M),X261V (e.g., N261V), X261W (e.g., N261W), X261Y (e.g., N261Y), X262C(e.g., L262C), X262E (e.g., L262E), X262Q (e.g., L262Q), and X274A(e.g., T274A), wherein each position corresponds to the position of thepolypeptide of SEQ ID NO: 2.

In an embodiment, the subtilase variant is selected from the groupconsisting of:

*99aE+A194P

N76D+Y167A+R170S+A194P N76D+Y167A+R170S+A194P+A228V+A230V

*99aE+S256D

L21D+*99aE N62D+Q245R+R170S R170L+Q206E+S57P A133P+Y167A+R170S+A194PS141N+Y167A+R170S+A194P Y167A+R170N Y167A+R170S+A172EN62D+Y167A+R170S+A194P N62D+R170S N62D+R170L

*97aN+A98T+S99D*98aA+S99D+N261D+L262Q

Q59D+N76D+Y167A+R170S+A194P Q59D+*99aE+Y209W+L262EQ59D+Y167A+R170S+A194P+Y209W+L262E Q59D+Y167A+R170S+A194P+L262EN62D+S101H+R170S+Y209W+L262E N62D+V104T+S156D+R170S+Y209W+L262EN62D+V104T+R170S+Y209W+L262E N62D+S156D+S163G+Y209W+Q245R+N248D+L262EN62D+S156D+S163G+Y209W+L262E N62D+S156D+S163K+Y209W+Q245R+N248D+L262EN62D+S156D+R170S+Y209W+L262E N62D+R170S+Y209W+Q245R+N248D+L262EN62D+R170S+Y209W+L262E N62D+R170S+N238E+L262EN76D+Y167A+R170S+A194P+N238E

*99aE+S101H+H120D+S163G+N261D*99aE+S156D+Y209W+L262E*99aE+B194P+G195E+Y209W+L262E*99aE+B194P+G195E+L262E*99aE+N238E+L262E

V104T+H120D+S163G+Y167A+R170S+A194P+N261DV104T+S156D+Y167A+R170S+A194P+Y209W+L262EV104T+Y167A+R170S+A194P+Y209W+N238E+L262EV104T+Y167A+R170S+A194P+N238E+L262E.

The subtilase variants may consist of 150 to 350, e.g., 175 to 330, 200to 310, 220 to 300, 240 to 290, 260 to 280 or 269, 270, 271, 272, 273,274 or 275 amino acids.

In one embodiment, the subtilase variant has improved stability, inparticular improved storage stability, compared to the parent subtilase.In a preferred embodiment, the subtilase variant has improved stability,in particular improved storage stability, and on par or improved washperformance compared to the parent subtilase.

In one embodiment, the subtilase variant has improved stability, inparticular improved wash stability, compared to the parent subtilase. Ina preferred embodiment, the subtilase variant has improved stability, inparticular improved in storage stability, and on par or improved washperformance compared to the parent subtilase.

In an embodiment, the subtilase variant has improved stability, inparticular improved in wash stability, and on par or improved washperformance compared to the parent subtilase wherein wash stability ismeasured using the ‘in wash stability assay’ and wash performance ismeasured using the Automatic Mechanical Stress Assay (AMSA) as describedin Example 2.

Parent Protease

The parent or the precursor protease may be any subtilase or even morepreferred any subtilisin as defined below.

Enzymes cleaving the amide linkages in protein substrates are classifiedas proteases, or (interchangeably) peptidases (see Walsh, 1979,Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco,Chapter 3).

Serine Proteases

A serine protease is an enzyme which catalyzes the hydrolysis of peptidebonds, and in which there is an essential serine residue at the activesite (White, Handler and Smith, 1973 “Principles of Biochemistry,” FifthEdition, McGraw-Hill Book Company, NY, pp. 271-272).

The bacterial serine proteases have molecular weights in the 20,000 to45,000 Dalton range. They are inhibited by diisopropylfluorophosphate.They hydrolyze simple terminal esters and are similar in activity toeukaryotic chymotrypsin, also a serine protease. A more narrow term,alkaline protease, covering a sub-group, reflects the high pH optimum ofsome of the serine proteases, from pH 9.0 to 11.0 (for review, seePriest, 1977, Bacteriological Rev. 41: 711-753).

Subtilases

A sub-group of the serine proteases tentatively designated subtilaseshas been proposed by Siezen et al., 1991, Protein Eng. 4:719-737 andSiezen et al., 1997, Protein Science 6:501-523. They are defined byhomology analysis of more than 170 amino acid sequences of serineproteases previously referred to as subtilisin-like proteases. Asubtilisin was previously often defined as a serine protease produced byGram-positive bacteria or fungi, and according to Siezen et al. now is asubgroup of subtilases. A wide variety of subtilases have beenidentified, and the amino acid sequence of a number of subtilases hasbeen determined. For a more detailed description of such subtilases andtheir amino acid sequences reference is made to Siezen et al. (1997).

Subtilisins

A subgroup of subtilases is subtilisins which are serine proteases fromthe family S8, in particular from the subfamily S8A, as defined by theMEROPS database (merops.sanger.ac.uk/cgi-bin/famsum?family=SB).

Subtilisin BPN′ and subtilisin 309 have the MEROPS numbers S08.034 andS08.003, respectively.

The term “parent subtilase” describes a subtilase defined according toSiezen et al., 1997, Protein Science 6: 501-523. For further details seedescription of “Subtilases” above. A parent subtilase may also be asubtilase isolated from a natural source, wherein subsequentmodifications (such as replacement(s) of the amino acid side chain(s),substitution(s), deletion(s) and/or insertion(s)) have been made whileretaining the characteristic of a subtilase. Furthermore, a parentsubtilase may be a subtilase which has been prepared by the DNAshuffling technique, such as described by Ness et al., 1999, NatureBiotechnology, 17: 893-896.

Alternatively, the term “parent subtilase” may be termed “precursorsubtilase” and is used to describe the starting protease into whichmutations are made to obtain the variant of the invention. The parentsubtilase is preferably of the subtilisin subgroups.

One subgroup of the subtilases, I-S1 or “true” subtilisins, include the“classical” subtilisins, such as subtilisin 168 (BSS168), subtilisinBPN′, subtilisin Carlsberg (ALCALASE®, Novozymes A/S), and subtilisin DY(BSSDY). BPN′ is subtilisin BPN′ from B. amyloliquefaciens, SubtilisinBPN′ has the amino acid sequence of SEQ ID NO: 2. A further subgroup ofthe subtilases, I-S2 or high alkaline subtilisins, is recognized bySiezen et al. (supra). Sub-group I-S2 proteases are described as highlyalkaline subtilisins and include enzymes such as subtilisin PB92(BAALKP) (MAXACAL®, Genencor International Inc.), subtilisin 147(BLS147) (ESPERASE®, Novozymes A/S), alkaline elastase YaB (BSEYAB) andsubtilisin 309 (SAVINASE®, Novozymes A/S) having the amino acid sequenceSEQ ID NO: 1.

For reference, Table 1 below gives a list of some acronyms for varioussubtilases mentioned herein. For further acronyms, see Siezen et al.(1991 and 1997).

TABLE 1 Acronyms of various subtilases Organism Enzyme Acronym SequenceBacillus subtilis 168 subtilisin I168, apr BSS168 SEQ ID NO: 3 Bacillussubtilisin BPN′ BASBPN SEQ ID NO: 2 amyloliquefaciens (NOVO) Bacillussubtilis DY subtilisin DY BSSDY SEQ ID NO: 4 Bacillus licheniformissubtilisin Carlsberg BLSCAR SEQ ID NO: 5 Bacillus lentus subtilisin 309BLSAVI SEQ ID NO: 1 Bacillus lentus subtilisin 147 BLS147 SEQ ID NO: 6Bacillus alcalophilus subtilisin PB92 BAPB92 SEQ ID NO: 7 PB92 BacillusYaB alkaline elastase YaB BYSYAB SEQ ID NO: 8 Bacillus sp. NKS-21subtilisin ALP I BSAPRQ SEQ ID NO: 9 Bacillus sp. G-825-6 subtilisinSendai BSAPRS SEQ ID NO: 10 Thermoactinomyces Thermitase TVTHER SEQ IDNO: 11 vulgaris

Homologous Subtilase Sequences

The homology between two amino acid sequences is in this contextdescribed by the parameter “identity” for purposes of the presentinvention, the degree of identity between two amino acid sequences isdetermined using the Needleman-Wunsch algorithm as described above. Theoutput from the routine is besides the amino acid alignment thecalculation of the “Percent Identity” between the two sequences.

Based on this description it is routine for a person skilled in the artto identify suitable homologous subtilases, which can be modifiedaccording to the invention.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 1, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 1. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 1.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 2, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 2. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 2.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 3, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 3. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 3.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 4, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 4. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 4.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 5, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 5. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 5.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 6, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 6. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 6.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 7, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 7. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 7.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 8, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 8. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 8.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 9, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 9. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 9.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 10, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 10. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 10.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 11, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 11. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 11.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent subtilase may be obtained from microorganisms of any genus.For purposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial protease. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces protease, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma protease.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis protease Strains of these species are readily accessibleto the public in a number of culture collections, such as the AmericanType Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures(CBS), and Agricultural Research Service Patent Culture Collection,Northern Regional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to a method for producing a subtilasevariant, comprising

(a) introducing into a parent subtilase a set of alterations selectedfrom the group consisting of:

(1) X167A+R170S+A194P (e.g., Y167A+R170S+A194P) and one or moresubstitutions selected from the group consisting of X59D (e.g., Q59D),X62D (e.g., N62D), X76D (e.g., N76D), X104T (e.g., V104T), X120D (e.g.,H120D), X133P (e.g. A133P), X141N (e.g. S141N), X156D (e.g., S156D),X163G (e.g., S163G), X209W (e.g., Y209W), X228V (e.g. A228V), X230V(e.g. A230V), X238E (e.g., N238E), X261D (e.g., N261D), and X262E (e.g.,L262E);

(2) 99aE and one or more substitutions selected from the groupconsisting of X21D (e.g., L21D), X59D (e.g., Q59D), X101H (e.g., S101H),X120D (e.g., H120D), X156D (e.g., S156D), X163G (e.g., S163G), X194P(e.g., A194P), X195E (e.g., G195E), X209W (e.g., Y209W), X238E (e.g.,N238E), X256D (e.g. N256D), X261D (e.g., N261D), and X262E (e.g.,L262E);

(3) X62D (e.g., N62D) and one or more substitutions selected from thegroup consisting of X101H (e.g., S101H), X104T (e.g., V104T), X156D(e.g., S156D), X163G (e.g., S163G), X170S,L (e.g., R170S,L), X209W(e.g., Y209W), X238E (e.g., N238E), X245R (e.g. Q245R) and X262E (e.g.,L262E);

(4) X62D+X245R+X248D (e.g., N62D+Q245R+N248D) and one or moresubstitutions selected from the group consisting of X156D (e.g., S156D),X163G (e.g., S163G), X163K (e.g., S163K), X170S (e.g., R170S), X209W(e.g., Y209W), and X262E (e.g., L262E);

(5) X170L, X170N or X170S (e.g. R170L, R170N, R170S) and one or moresubstitutions selected from the group consisting of X57P (e.g. S57P),X167A (e.g. Y167A), X172E (e.g. A172E), X206E (e.g. Q206E),

(6) X99D (e.g. S99D) and one or more substitutions selected from thegroup consisting of *97aN, *98aA, X98T (e.g. A98T), X261D (e.g., N261D),and X262Q (e.g., L262Q);

wherein

-   -   (i) the positions correspond to the positions of the polypeptide        of SEQ ID NO: 2;    -   (ii) the variant has protease activity; and    -   (iii) the variant has at least 60%, e.g., at least 65%, at least        70%, at least 75%, at least 80%, at least 85%, at least 90%, at        least 95%, at least 96%, at least 97%, at least 98% but less        than 100% sequence identity to the polypeptide of SEQ ID NO: 1,        2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; and

(b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., US 2004/0171154; Storici et al., 2001,Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to polynucleotides encoding a variantof the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that when introduced into the hostcell is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants with protease activity. These detectionmethods include, but are not limited to, use of specific antibodies,formation of an enzyme product, or disappearance of an enzyme substrate.For example, an enzyme assay may be used to determine the activity ofthe variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

The subtilase variant of the present invention may be incorporated in acomposition such as a detergent composition.

The choice of additional components is within the skill of the artisanand includes conventional ingredients, including the exemplarynon-limiting components set forth below. The choice of components mayinclude, for fabric care, the consideration of the type of fabric to becleaned, the type and/or degree of soiling, the temperature at whichcleaning is to take place, and the formulation of the detergent product.Although components mentioned below are categorized by general headeraccording to a particular functionality, this is not to be construed asa limitation, as a component may comprise additional functionalities aswill be appreciated by the person skilled in the art.

In a particular embodiment, a detergent composition may comprise asubtilase variant of the invention and one or more detergent components,such as surfactants, hydrotropes, builders, co-builders, chelators orchelating agents, bleaching system or bleach components, polymers,fabric hueing agents, fabric conditioners, foam boosters, sudssuppressors, dispersants, dye transfer inhibitors, fluorescent whiteningagents, perfume, optical brighteners, bactericides, fungicides, soilsuspending agents, soil release polymers, anti-redeposition agents,enzyme inhibitors or stabilizers, enzyme activators, antioxidants, andsolubilizers.

In one embodiment of the present invention, the subtilase variant of thepresent invention may be added to a detergent composition in an amountcorresponding to 0.01-200 mg of enzyme protein per liter of wash liquor,preferably 0.05-50 mg of enzyme protein per liter of wash liquor, inparticular 0.1-10 mg of enzyme protein per liter of wash liquor.

A composition for use in automatic dishwash (ADVV), for example, mayinclude 0.0001%-50%, such as 0.001%-30%, such as 0.01%-20%, such as0.5-15% of enzyme protein by weight of the composition.

A composition for use in laundry granulation, for example, may include0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as 0.05%-5% ofenzyme protein by weight of the composition.

A composition for use in laundry liquid, for example, may include0.0001%-10%, such as 0.001-7%, such as 0.1%-5% of enzyme protein byweight of the composition.

The enzymes such as the subtilase variant of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example, WO92/19709 and WO 92/19708 or the variants according to the invention maybe stabilized using peptide aldehydes or ketones such as described in WO2005/105826 and WO 2009/118375.

A subtilase variant of the present invention may also be incorporated inthe detergent formulations disclosed in WO 97/07202, which is herebyincorporated by reference.

Surfactants

The detergent composition may comprise one or more surfactants, whichmay be anionic and/or cationic and/or non-ionic and/or semi-polar and/orzwitterionic, or a mixture thereof. In a particular embodiment, thedetergent composition includes a mixture of one or more nonionicsurfactants and one or more anionic surfactants. The surfactant(s) istypically present at a level of from about 0.1% to 60% by weight, suchas about 1% to about 40%, or about 3% to about 20%, or about 3% to about10%. The surfactant(s) is chosen based on the desired cleaningapplication, and includes any conventional surfactant(s) known in theart. Any surfactant known in the art for use in detergents may beutilized. Surfactants lower the surface tension in the detergent, whichallows the stain being cleaned to be lifted and dispersed and thenwashed away.

When included therein the detergent will usually contain from about 1%to about 40% by weight, such as from about 5% to about 30%, includingfrom about 5% to about 15%, or from about 20% to about 25% of an anionicsurfactant. Non-limiting examples of anionic surfactants includesulfates and sulfonates, in particular, linear alkylbenzenesulfonates(LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES, also known as alcoholethoxysulfates or fatty alcohol ether sulfates), secondaryalkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methylesters (alpha-SFMe or SES) including methyl ester sulfonate (MES),alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid(DTSA), fatty acid derivatives of amino acids, diesters and monoestersof sulfo-succinic acid or soap, and combinations thereof.

When included therein the detergent will usually contain from about 0%to about 10% by weight of a cationic surfactant. Non-limiting examplesof cationic surfactants include alklydimethylethanolamine quat (ADMEAQ),cetyltrimethylammonium bromide (CTAB), dimethyldistearylammoniumchloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternaryammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, andcombinations thereof.

When included therein the detergent will usually contain from about 0.2%to about 40% by weight of a non-ionic surfactant, for example from about0.5% to about 30%, in particular from about 1% to about 20%, from about3% to about 10%, such as from about 3% to about 5%, or from about 8% toabout 12%. Non-limiting examples of non-ionic surfactants includealcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylatedfatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such asethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenolethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides(APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fattyacid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides(EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glucamides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

When included therein the detergent will usually contain from about 0%to about 10% by weight of a semipolar surfactant. Non-limiting examplesof semipolar surfactants include amine oxides (AO) such asalkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide andN-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acidalkanolamides and ethoxylated fatty acid alkanolamides, and combinationsthereof.

When included therein the detergent will usually contain from about 0%to about 10% by weight of a zwitterionic surfactant. Non-limitingexamples of zwitterionic surfactants include betaine,alkyldimethylbetaine, sulfobetaine, and combinations thereof.

Hydrotropes

A hydrotrope is a compound that solubilizes hydrophobic compounds inaqueous solutions (or oppositely, polar substances in a non-polarenvironment). Typically, hydrotropes have both hydrophilic andhydrophobic characters (so-called amphiphilic properties as known fromsurfactants); however the molecular structure of hydrotropes generallydo not favor spontaneous self-aggregation, see, e.g., review by Hodgdonand Kaler, 2007, Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmiceller, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behavior, stability, and colloidal properties of systemscontaining substances of polar and non-polar character, includingmixtures of water, oil, surfactants, and polymers. Hydrotropes areclassically used across industries from pharma, personal care, food, totechnical applications. Use of hydrotropes in detergent compositionsallows for example more concentrated formulations of surfactants (as inthe process of compacting liquid detergents by removing water) withoutinducing undesired phenomena such as phase separation or high viscosity.

The detergent may contain 0-5% by weight, such as about 0.5 to about 5%,or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in theart for use in detergents may be utilized. Non-limiting examples ofhydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate(STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS),sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers,sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodiumethylhexyl sulfate, and combinations thereof.

Builders and Co-Builders

The detergent composition may contain about 0-65% by weight, such asabout 5% to about 45% of a detergent builder or co-builder, or a mixturethereof. In a dish wash detergent, the level of builder is typically40-65%, particularly 50-65%. Builders and chelators soften, e.g., thewash water by removing the metal ions form the liquid. The builderand/or co-builder may particularly be a chelating agent that formswater-soluble complexes with Ca and Mg. Any builder and/or co-builderknown in the art for use in laundry detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, alsoknown as iminodiethanol), triethanolamine (TEA, also known as2,2′,2″-nitrilotriethanol), and carboxymethyl inulin (CMI), andcombinations thereof.

The detergent composition may also contain 0-20% by weight, such asabout 5% to about 10%, of a detergent co-builder, or a mixture thereof.The detergent composition may include a co-builder alone, or incombination with a builder, for example a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid(HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA),diethylenetriaminepentakis (methylenephosphonic acid) (DTPMPA or DTMPA),N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoaceticacid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), asparticacid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid(SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL),N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid(MIDA), α-alanine-N, N-diacetic acid (α-ALDA), serine-N, N-diacetic acid(SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N, N-diaceticacid (PHDA), anthranilic acid-N, N-diacetic acid (ANDA), sulfanilicacid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) andsulfomethyl-N, N-diacetic acid (SMDA),N-(2-hydroxyethyl)-ethylidenediamine-N, N′,N′-triacetate (HEDTA),diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonicacid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), andcombinations and salts thereof. Further exemplary builders and/orco-builders are described in, e.g., WO 2009/102854 and U.S. Pat. No.5,977,053

Bleaching Systems

The detergent may contain 0-50% by weight, such as about 0.1% to about25%, of a bleaching system. Bleach systems remove discolor often byoxidation, and many bleaches also have strong bactericidal properties,and are used for disinfecting and sterilizing. Any bleaching systemknown in the art for use in laundry detergents may be utilized. Suitablebleaching system components include bleaching catalysts, photobleaches,bleach activators, sources of hydrogen peroxide such as sodiumpercarbonate and sodium perborates, preformed peracids and mixturesthereof. Suitable preformed peracids include, but are not limited to,peroxycarboxylic acids and salts, percarbonic acids and salts, perimidicacids and salts, peroxymonosulfuric acids and salts, for example, Oxone(R), and mixtures thereof. Non-limiting examples of bleaching systemsinclude peroxide-based bleaching systems, which may comprise, forexample, an inorganic salt, including alkali metal salts such as sodiumsalts of perborate (usually mono- or tetra-hydrate), percarbonate,persulfate, perphosphate, persilicate salts, in combination with aperacid-forming bleach activator. The term bleach activator is meantherein as a compound which reacts with peroxygen bleach like hydrogenperoxide to form a peracid. The peracid thus formed constitutes theactivated bleach. Suitable bleach activators to be used herein includethose belonging to the class of esters amides, imides or anhydrides.Suitable examples are tetracetylethylene diamine (TAED), sodium4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxydodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS),4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed in WO98/17767. A particular family of bleach activators of interest wasdisclosed in EP 624154 and particularly preferred in that family isacetyl triethyl citrate (ATC). ATC or a short chain triglyceride liketriacetin has the advantage that it is environmental friendly as iteventually degrades into citric acid and alcohol. Furthermore acetyltriethyl citrate and triacetin has a good hydrolytical stability in theproduct upon storage and it is an efficient bleach activator. FinallyATC provides a good building capacity to the laundry additive.Alternatively, the bleaching system may comprise peroxyacids of, forexample, the amide, imide, or sulfone type. The bleaching system mayalso comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP).The bleaching system may also include a bleach catalyst. In someembodiments the bleach component may be an organic catalyst selectedfrom the group consisting of organic catalysts having the followingformula:

(iii) and mixtures thereof; wherein each R¹ is independently a branchedalkyl group containing from 9 to 24 carbons or linear alkyl groupcontaining from 11 to 24 carbons, preferably each R¹ is independently abranched alkyl group containing from 9 to 18 carbons or linear alkylgroup containing from 11 to 18 carbons, more preferably each R¹ isindependently selected from the group consisting of 2-propylheptyl,2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl,n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl andiso-pentadecyl. Other exemplary bleaching systems are described, e.g.,in WO 2007/087258, WO 2007/087244, WO 2007/087259 and WO 2007/087242.Suitable photobleaches may for example be sulfonated zincphthalocyanine.

Polymers

The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2%or 0.2-1% of a polymer. Any polymer known in the art for use indetergents may be utilized. The polymer may function as a co-builder asmentioned above, or may provide antiredeposition, fiber protection, soilrelease, dye transfer inhibition, grease cleaning and/or anti-foamingproperties. Some polymers may have more than one of the above-mentionedproperties and/or more than one of the below-mentioned motifs. Exemplarypolymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol)(PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) orpoly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine),carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA,poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers,hydrophobically modified CMC (HM-CMC) and silicones, copolymers ofterephthalic acid and oligomeric glycols, copolymers of poly(ethyleneterephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP,poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO)and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplarypolymers include sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

Fabric Hueing Agents

The detergent compositions may also include fabric hueing agents such asdyes or pigments, which when formulated in detergent compositions candeposit onto a fabric when the fabric is contacted with a wash liquorcomprising the detergent compositions and thus altering the tint of thefabric through absorption/reflection of visible light. Fluorescentwhitening agents emit at least some visible light. In contrast, fabrichueing agents alter the tint of a surface as they absorb at least aportion of the visible light spectrum. Suitable fabric hueing agentsinclude dyes and dye-clay conjugates, and may also include pigments.Suitable dyes include small molecule dyes and polymeric dyes. Suitablesmall molecule dyes include small molecule dyes selected from the groupconsisting of dyes falling into the Colour Index (C.I.) classificationsof Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, AcidViolet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, forexample as described in WO 2005/003274, WO 2005/003275, WO 2005/003276and EP 1876226 (hereby incorporated by reference). The detergentcomposition preferably comprises from about 0.00003 wt. % to about 0.2wt. %, from about 0.00008 wt. % to about 0.05 wt. %, or even from about0.0001 wt. % to about 0.04 wt. % fabric hueing agent. The compositionmay comprise from 0.0001 wt % to 0.2 wt. % fabric hueing agent, this maybe especially preferred when the composition is in the form of a unitdose pouch. Suitable hueing agents are also disclosed in, e.g., WO2007/087257 and WO 2007/087243.

Additional Enzymes

The detergent additive as well as the detergent composition may compriseone or more (additional) enzymes such as an amylase, arabinase,carbohydrase, cellulase (e.g., endoglucanase), cutinase, galactanase,haloperoxygenase, lipase, mannanase, oxidase, e.g., laccase and/orperoxidase, pectinase, pectin lyases, protease, xylanase, xanthanase,and xyloglucanase.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 495257, EP 531372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 531315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Examples of cellulases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4) are described in WO 02/99091.

Other examples of cellulases include the family 45 cellulases describedin WO 96/29397, and especially variants thereof having substitution,insertion and/or deletion at one or more of the positions correspondingto the following positions in SEQ ID NO: 8 of WO 02/99091: 2, 4, 7, 8,10, 13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43,44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82,84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113,114, 117, 119, 121, 133, 136, 137, 138, 139, 140a, 141, 143a, 145, 146,147, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c,160e, 160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178,181, 183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and/or 20,preferably selected among P19A, G20K, Q44K, N48E, Q119H or Q146 R.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases

The composition may comprise one or more additional proteases includingthose of bacterial, fungal, plant, viral or animal origin, e.g.,vegetable or microbial origin. Microbial origin is preferred. Chemicallymodified or protein engineered mutants are included. It may be analkaline protease, such as a serine protease or a metalloprotease. Aserine protease may for example be of the 51 family, such as trypsin, orthe S8 family such as subtilisin. A metalloproteases protease may forexample be a thermolysin from, e.g., family M4 or other metalloproteasesuch as those from M5, M7 or M8 families.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO 2007/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens.

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®,Purafect MAO, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®,FN3®, FN4®, Excellase®, Eraser®, Opticlean® and Optimase®(Danisco/DuPont), Axapem™ (Gist-Brocades N.V.), BLAP (sequence shown inFIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (Henkel AG) andKAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and Cutinases

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g., from T.lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP 305216, cutinase from Humicola, e.g., H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamedto Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95/06720 & WO96/27002), P. wisconsinensis (WO 96/12012), GDSL-type Streptomyceslipases (WO 2010/065455), cutinase from Magnaporthe grisea (WO2010/107560), cutinase from Pseudomonas mendocina (U.S. Pat. No.5,389,536), lipase from Thermobifida fusca (WO 2011/084412), Geobacillusstearothermophilus lipase (WO 2011/084417), lipase from Bacillussubtilis (WO 2011/084599), and lipase from Streptomyces griseus (WO2011/150157) and S. pristinaespiralis (WO 2012/137147).

Other examples are lipase variants such as those described in EP 407225,WO 92/05249, WO 94/01541, WO 94/25578, WO 95/14783, WO 95/30744, WO95/35381, WO 95/22615, WO 96/00292, WO 97/04079, WO 97/07202, WO00/34450, WO 00/60063, WO 01/92502, WO 2007/87508 and WO 2009/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g., acyltransferases with homologyto Candida antarctica lipase A (WO 2010/111143), acyltransferase fromMycobacterium smegmatis (WO 2005/056782), perhydrolases from the CE 7family (WO 2009/067279), and variants of the M. smegmatis perhydrolasein particular the S54V variant used in the commercial product GentlePower Bleach from Huntsman Textile Effects Pte Ltd (WO 2010/100028).

Amylases

Suitable amylases which can be used together with the subtilase variantsof the invention may be an alpha-amylase or a glucoamylase and may be ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB 1,296,839.

Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQID NO: 4 of WO 99/19467, such as variants with substitutions in one ormore of the following positions: 15, 23, 105, 106, 124, 128, 133, 154,156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243,264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO02/10355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having thesubstitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Other suitable amylases are amylases having the sequence of SEQ ID NO: 6in WO 99/19467 or variants thereof having 90% sequence identity to SEQID NO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, 1206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/23873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQID 2 of WO 96/23873 for numbering. More preferred variants are thosehaving a deletion in two positions selected from 181, 182, 183 and 184,such as 181 and 182, 182 and 183, or positions 183 and 184. Mostpreferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7are those having a deletion in positions 183 and 184 and a substitutionin one or more of positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO2008/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 2008/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ IDNO: 10 in WO 01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO2009/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K;

S125A+N128C+K178L+T182G+Y305R+G475K;

N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

N128C+K178L+T182G+Y305R+G475K;

wherein the variants are C-terminally truncated and optionally furthercomprise a substitution at position 243 and/or a deletion at position180 and/or position 181.

Further suitable amylases are amylases having SEQ ID NO: 1 of WO2013/184577 or variants having 90% sequence identity to SEQ ID NO: 1thereof. Preferred variants of SEQ ID NO: 1 are those having asubstitution, a deletion or an insertion in one of more of the followingpositions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241,R458, T459, D460, G476 and G477. More preferred variants of SEQ ID NO: 1are those having the substitution in one of more of the followingpositions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S,D460T, G476K and G477K and/or a deletion in position R178 and/or S179 orof T180 and/or G181. Most preferred amylase variants of SEQ ID NO: 1comprise the substitutions:

E187P+I203Y+R458N+T459S+D460T+G476K

E187P+I203Y+G476K

and optionally further comprise a substitution at position 241 and/or adeletion at position 178 and/or position 179.

Further suitable amylases are amylases having SEQ ID NO: 1 of WO2010/104675 or variants having 90% sequence identity to SEQ ID NO: 1thereof. Preferred variants of SEQ ID NO: 1 are those having asubstitution, a deletion or an insertion in one of more of the followingpositions: N21, D97, V128 K177, R179, S180, I181, G182, M200, L204,E242, G477 and G478. More preferred variants of SEQ ID NO: 1 are thosehaving the substitution in one of more of the following positions: N21D,D97N, V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or adeletion in position R179 and/or S180 or of I181 and/or G182. Mostpreferred amylase variants of SEQ ID NO: 1 comprise the substitutionsN21D+D97N+V128I, and optionally further comprise a substitution atposition 200 and/or a deletion at position 180 and/or position 181.Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO 01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described in WO2011/098531, WO 2013/001078 and WO 2013/001087. Commercially availableamylases are Duramyl™, Termamyl™, Fungamyr™, Stainzyme™, StainzymePlus™, Natalase™, Liquozyme X and BAN™ (from Novozymes A/S), andRapidase™, Purastar™/Effectenz™, Powerase, Preferenz S1000, PreferenzS100 and Preferenz S110 (from Genencor International Inc./DuPont).

Peroxidases/Oxidases

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,i.e., a separate additive or a combined additive, can be formulated, forexample, as a granulate, liquid, slurry, etc. Preferred detergentadditive formulations are granulates, in particular non-dustinggranulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

Adjunct Materials

Any detergent components known in the art for use in laundry detergentsmay also be utilized. Other optional detergent components includeanti-corrosion agents, anti-shrink agents, anti-soil redepositionagents, anti-wrinkling agents, bactericides, binders, corrosioninhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in laundry detergents may be utilized. The choice ofsuch ingredients is well within the skill of the artisan.

Dispersants: The detergent compositions can also contain dispersants. Inparticular powdered detergents may comprise dispersants. Suitablewater-soluble organic materials include the homo- or co-polymeric acidsor their salts, in which the polycarboxylic acid comprises at least twocarboxyl radicals separated from each other by not more than two carbonatoms. Suitable dispersants are for example described in PowderedDetergents, Surfactant science series volume 71, Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents: The detergent compositions may alsoinclude one or more dye transfer inhibiting agents. Suitable polymericdye transfer inhibiting agents include, but are not limited to,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles or mixtures thereof. When present in a subjectcomposition, the dye transfer inhibiting agents may be present at levelsfrom about 0.0001% to about 10%, from about 0.01% to about 5% or evenfrom about 0.1% to about 3% by weight of the composition.

Fluorescent whitening agent: The detergent compositions will preferablyalso contain additional components that may tint articles being cleaned,such as fluorescent whitening agent or optical brighteners. Wherepresent the brightener is preferably at a level of about 0.01% to about05%. Any fluorescent whitening agent suitable for use in a laundrydetergent composition may be used in the composition of the presentinvention. The most commonly used fluorescent whitening agents are thosebelonging to the classes of diaminostilbene-sulphonic acid derivatives,diarylpyrazoline derivatives and bisphenyl-distyryl derivatives.Examples of the diaminostilbene-sulphonic acid derivative type offluorescent whitening agents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2′-disulphonate;4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate and2-(stilbyl-4″-naptho-1,2′:4,5)-1,2,3-trizole-2″-sulphonate. Preferredfluorescent whitening agents are Tinopal DMS and Tinopal CBS availablefrom Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium saltof 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbenedisulphonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use include the 1-3-diaryl pyrazolines and the7-alkylaminocoumarins. Suitable fluorescent brightener levels includelower levels of from about 0.01, from 0.05, from about 0.1 or even fromabout 0.2 wt. % to upper levels of 0.5 or even 0.75 wt. %.

Soil release polymers: The detergent compositions may also include oneor more soil release polymers which aid the removal of soils fromfabrics such as cotton and polyester based fabrics, in particular theremoval of hydrophobic soils from polyester based fabrics. The soilrelease polymers may for example be nonionic or anionic terephthaltebased polymers, polyvinyl caprolactam and related copolymers, vinylgraft copolymers, polyester polyamides see for example Chapter 7 inPowdered Detergents, Surfactant science series volume 71, Marcel Dekker,Inc. Another type of soil release polymers are amphiphilic alkoxylatedgrease cleaning polymers comprising a core structure and a plurality ofalkoxylate groups attached to that core structure. The core structuremay comprise a polyalkylenimine structure or a polyalkanolaminestructure as described in detail in WO 2009/087523 (hereby incorporatedby reference). Furthermore random graft co-polymers are suitable soilrelease polymers Suitable graft co-polymers are described in more detailin WO 2007/138054, WO 2006/108856 and WO 2006/113314 (herebyincorporated by reference). Other soil release polymers are substitutedpolysaccharide structures especially substituted cellulosic structuressuch as modified cellulose deriviatives such as those described in EP1867808 or WO 03/040279 (both are hereby incorporated by reference).Suitable cellulosic polymers include cellulose, cellulose ethers,cellulose esters, cellulose amides and mixtures thereof. Suitablecellulosic polymers include anionically modified cellulose, nonionicallymodified cellulose, cationically modified cellulose, zwitterionicallymodified cellulose, and mixtures thereof. Suitable cellulosic polymersinclude methyl cellulose, carboxy methyl cellulose, ethyl cellulose,hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, estercarboxy methyl cellulose, and mixtures thereof.

Anti-redeposition agents: The detergent compositions may also includeone or more anti-redeposition agents such as carboxymethylcellulose(CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP),polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylicacid, copolymers of acrylic acid and maleic acid, and ethoxylatedpolyethyleneimines. The cellulose based polymers described under soilrelease polymers above may also function as anti-redeposition agents.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,and structurants for liquid detergents and/or structure elasticizingagents.

Formulation of Detergent Products

The detergent composition may be in any convenient form, e.g., a bar, ahomogenous tablet, a tablet having two or more layers, a pouch havingone or more compartments, a regular or compact powder, a granule, apaste, a gel, or a regular, compact or concentrated liquid. There are anumber of detergent formulation forms such as layers (same or differentphases), pouches, as well as forms for machine dosing unit.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g., without allowing the release of the composition from the pouchprior to water contact. The pouch is made from water soluble film whichencloses an inner volume. The inner volume can be divided intocompartments of the pouch. Preferred films are polymeric materialspreferably polymers which are formed into a film or sheet. Preferredpolymers, copolymers or derivates thereof are selected polyacrylates,and water soluble acrylate copolymers, methyl cellulose, carboxy methylcellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, mostpreferably polyvinyl alcohol copolymers and, hydroxypropyl methylcellulose (HPMC). Preferably the level of polymer in the film forexample PVA is at least about 60%. Preferred average molecular weightwill typically be about 20,000 to about 150,000. Films can also be ofblend compositions comprising hydrolytically degradable and watersoluble polymer blends such as polylactide and polyvinyl alcohol (knownunder the Trade reference M8630 as sold by Chris Craft In. Prod. ofGary, Ind., US) plus plasticisers like glycerol, ethylene glycerol,Propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry detergent composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids. See, e.g., US2009/0011970.

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent. A liquid or geldetergent may be non-aqueous.

Laundry Soap Bars

The subtilase variants of the invention may be added to laundry soapbars and used for hand washing laundry, fabrics and/or textiles. Theterm laundry soap bar includes laundry bars, soap bars, combo bars,syndet bars and detergent bars. The types of bar usually differ in thetype of surfactant they contain, and the term laundry soap bar includesthose containing soaps from fatty acids and/or synthetic soaps. Thelaundry soap bar has a physical form which is solid and not a liquid,gel or a powder at room temperature. The term solid is defined as aphysical form which does not significantly change over time, i.e., if asolid object (e.g., laundry soap bar) is placed inside a container, thesolid object does not change to fill the container it is placed in. Thebar is a solid typically in bar form but can be in other solid shapessuch as round or oval.

The laundry soap bar may contain one or more additional enzymes,protease inhibitors such as peptide aldehydes (or hydrosulfite adduct orhemiacetal adduct), boric acid, borate, borax and/or phenylboronic acidderivatives such as 4-formylphenylboronic acid, one or more soaps orsynthetic surfactants, polyols such as glycerine, pH controllingcompounds such as fatty acids, citric acid, acetic acid and/or formicacid, and/or a salt of a monovalent cation and an organic anion whereinthe monovalent cation may be for example Na⁺, K⁺ or NH₄ ⁺ and theorganic anion may be for example formate, acetate, citrate or lactatesuch that the salt of a monovalent cation and an organic anion may be,for example, sodium formate.

The laundry soap bar may also contain complexing agents like EDTA andHEDP, perfumes and/or different type of fillers, surfactants, e.g.,anionic synthetic surfactants, builders, polymeric soil release agents,detergent chelators, stabilizing agents, fillers, dyes, colorants, dyetransfer inhibitors, alkoxylated polycarbonates, suds suppressers,structurants, binders, leaching agents, bleaching activators, clay soilremoval agents, anti-redeposition agents, polymeric dispersing agents,brighteners, fabric softeners, perfumes and/or other compounds known inthe art.

The laundry soap bar may be processed in conventional laundry soap barmaking equipment such as but not limited to: mixers, plodders, e.g., atwo stage vacuum plodder, extruders, cutters, logo-stampers, coolingtunnels and wrappers. The process is not limited to preparing thelaundry soap bars by any single method. The premix may be added to thesoap at different stages of the process. For example, the premixcontaining a soap, an enzyme, optionally one or more additional enzymes,a protease inhibitor, and a salt of a monovalent cation and an organicanion may be prepared and the mixture is then plodded. The enzyme andoptional additional enzymes may be added at the same time as theprotease inhibitor for example in liquid form. Besides the mixing stepand the plodding step, the process may further comprise the steps ofmilling, extruding, cutting, stamping, cooling and/or wrapping.

Granular Detergent Formulations

A granular detergent may be formulated as described in WO 2009/092699,EP 1705241, EP 1382668, WO 2007/001262, U.S. Pat. No. 6,472,364, WO2004/074419 or WO 2009/102854. Other detergent formulations aredescribed in WO 2009/124162, WO 2009/124163, WO 2009/117340, WO2009/117341, WO 2009/117342, WO 2009/072069, WO 2009/063355, WO2009/132870, WO 2009/121757, WO 2009/112296, WO 2009/112298, WO2009/103822, WO 2009/087033, WO 2009/050026, WO 2009/047125, WO2009/047126, WO 2009/047127, WO 2009/047128, WO 2009/021784, WO2009/010375, WO 2009/000605, WO 2009/122125, WO 2009/095645, WO2009/040544, WO 2009/040545, WO 2009/024780, WO 2009/004295, WO2009/004294, WO 2009/121725, WO 2009/115391, WO 2009/115392, WO2009/074398, WO 2009/074403, WO 2009/068501, WO 2009/065770, WO2009/021813, WO 2009/030632, WO 2009/015951, WO 2011/025615, WO2011/016958, WO 2011/005803, WO 2011/005623, WO 2011/005730, WO2011/005844, WO 2011/005904, WO 2011/005630, WO 2011/005830, WO2011/005912, WO 2011/005905, WO 2011/005910, WO 2011/005813, WO2010/135238, WO 2010/120863, WO 2010/108002, WO 2010/111365, WO2010/108000, WO 2010/107635, WO 2010/090915, WO 2010/033976, WO2010/033746, WO 2010/033747, WO 2010/033897, WO 2010/033979, WO2010/030540, WO 2010/030541, WO 2010/030539, WO 2010/024467, WO2010/024469, WO 2010/024470, WO 2010/025161, WO 2010/014395, WO2010/044905, WO 2010/145887, WO 2010/142503, WO 2010/122051, WO2010/102861, WO 2010/099997, WO 2010/084039, WO 2010/076292, WO2010/069742, WO 2010/069718, WO 2010/069957, WO 2010/057784, WO2010/054986, WO 2010/018043, WO 2010/003783, WO 2010/003792, WO2011/023716, WO 2010/142539, WO 2010/118959, WO 2010/115813, WO2010/105942, WO 2010/105961, WO 2010/105962, WO 2010/094356, WO2010/084203, WO 2010/078979, WO 2010/072456, WO 2010/069905, WO2010/076165, WO 2010/072603, WO 2010/066486, WO 2010/066631, WO2010/066632, WO 2010/063689, WO 2010/060821, WO 2010/049187, WO2010/031607, and WO 2010/000636.

Uses

The subtilase variants according to the invention or compositionsthereof may be used in laundering of textile and fabrics, such as household laundry washing and industrial laundry washing.

The variants according to the invention or compositions thereof may alsobe used in cleaning hard surfaces such as floors, tables, walls, roofsetc. as well as surfaces of hard objects such as cars (car wash) anddishes (dish wash).

The subtilase variants of the present invention may be added to and thusbecome a component of a detergent composition. Thus the subtilasevariants of the invention may be used in a cleaning process such aslaundering and/or hard surface cleaning.

A detergent composition may be formulated, for example, as a hand ormachine laundry detergent composition including a laundry additivecomposition suitable for pre-treatment of stained fabrics and a rinseadded fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

A detergent additive may comprise a polypeptide of the present inventionas described herein.

A cleaning process or the textile care process may for example be alaundry process, a dishwashing process or cleaning of hard surfaces suchas bathroom tiles, floors, table tops, drains, sinks and washbasins.Laundry processes can for example be household laundering, but it mayalso be industrial laundering. A cleaning process includes a process forlaundering of fabrics and/or garments where the process comprisestreating fabrics with a washing solution containing a detergentcomposition, and at least one protease variant of the invention. Thecleaning process or a textile care process can for example be carriedout in a machine washing process or in a manual washing process. Thewashing solution can for example be an aqueous washing solutioncontaining a detergent composition.

The last few years there has been an increasing interest in replacingcomponents in detergents, which is derived from petrochemicals withrenewable biological components such as enzymes and polypeptides withoutcompromising the wash performance. When the components of detergentcompositions change new enzyme activities or new enzymes havingalternative and/or improved properties compared to the common useddetergent enzymes such as proteases, lipases and amylases is needed toachieve a similar or improved wash performance when compared to thetraditional detergent compositions.

The subtilase variants of the invention may be used in a proteinaceousstain removing processes. The proteinaceous stains may be stains suchas, e.g., baby food, sebum, cocoa, egg, blood, milk, ink, grass, or acombination thereof.

Typical detergent compositions include various components in addition tothe enzymes, these components have different effects, some componentslike the surfactants lower the surface tension in the detergent, whichallows the stain being cleaned to be lifted and dispersed and thenwashed away, other components like bleach systems remove discolor oftenby oxidation and many bleaches also have strong bactericidal properties,and are used for disinfecting and sterilizing. Yet other components likebuilder and chelator softens, e.g., the wash water by removing the metalions form the liquid.

A subtilase variant of the invention may be used in a compositioncomprising and one or more detergent components, such as surfactants,hydrotropes, builders, co-builders, chelators or chelating agents,bleaching system or bleach components, polymers, fabric hueing agents,fabric conditioners, foam boosters, suds suppressors, dispersants, dyetransfer inhibitors, fluorescent whitening agents, perfume, opticalbrighteners, bactericides, fungicides, soil suspending agents, soilrelease polymers, anti-redeposition agents, enzyme inhibitors orstabilizers, enzyme activators, antioxidants, and solubilizers.

A subtilase variant of the invention may be used in a compositioncomprising one or more additional enzymes selected from the groupcomprising of proteases, amylases, lipases, cutinases, cellulases,endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidase, haloperoxygenases, catalases and mannanases, or any mixturethereof.

In a particular a subtilase variant of the invention maybe used in acomposition comprising one or more additional enzymes selected from thegroup comprising of proteases, amylases, lipases, cutinases, cellulases,endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidase, haloperoxygenases, catalases and mannanases, or any mixturethereof and one or more detergent components, such as surfactants,hydrotropes, builders, co-builders, chelators or chelating agents,bleaching system or bleach components, polymers, fabric hueing agents,fabric conditioners, foam boosters, suds suppressors, dispersants, dyetransfer inhibitors, fluorescent whitening agents, perfume, opticalbrighteners, bactericides, fungicides, soil suspending agents, soilrelease polymers, anti-redeposition agents, enzyme inhibitors orstabilizers, enzyme activators, antioxidants, and solubilizers.

Washing Method

A protease variant of the invention may be used in a detergentcomposition in a method of cleaning a fabric, a dishware or hard surfacewith

A method of cleaning may comprise the steps of: contacting an objectwith a detergent composition comprising a protease variant of theinvention under conditions suitable for cleaning the object. Thedetergent composition may be used in a laundry or a dish wash process.

A method for removing stains from fabric or dishware may comprisecontacting the fabric or dishware with a composition comprising asubtilase variant of the invention under conditions suitable forcleaning the object.

The method includes treating fabrics (e.g., to desize a textile) usingone or more of the subtilase variants of the invention. The subtilasevariants can be used in any fabric-treating method which is well knownin the art (see, e.g., U.S. Pat. No. 6,077,316). For example, in oneaspect, the feel and appearance of a fabric is improved by a methodcomprising contacting the fabric with a protease in a solution. In oneaspect, the fabric is treated with the solution under pressure.

Detergent compositions are suited for use in laundry and hard surfaceapplications, including dish wash. Methods for laundering a fabric orwashing dishware may comprise the steps of contacting thefabric/dishware to be cleaned with a solution comprising the detergentcomposition. The fabric may comprise any fabric capable of beinglaundered in normal consumer use conditions. The dishware may compriseany dishware such as crockery, cutlery, ceramics, plastics such asmelamine, metals, china, glass and acrylics. The solution preferably hasa pH from about 5.5 to about 11.5. The compositions may be employed atconcentrations from about 100 ppm, preferably 500 ppm to about 15,000ppm in solution. The water temperatures typically range from about 5° C.to about 95° C., including about 10° C., about 15° C., about 20° C.,about 25° C., about 30° C., about 35° C., about 40° C., about 45° C.,about 50° C., about 55° C., about 60° C., about 65° C., about 70° C.,about 75° C., about 80° C., about 85° C. and about 90° C. The water tofabric ratio is typically from about 1:1 to about 30:1.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents and protease inhibitors, e.g., a polyolsuch as propylene glycol or glycerol, a sugar or sugar alcohol,different salts such as NaCl; KCl; lactic acid, formic acid, boric acid,or a boric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, or apeptide aldehyde such as di-, tri- or tetrapeptide aldehydes or aldehydeanalogues (either of the form B1-B0-R wherein, R is H, CH3, CX3, CHX2,or CH2X (X=halogen), B0 is a single amino acid residue (preferably withan optionally substituted aliphatic or aromatic side chain); and B1consists of one or more amino acid residues (preferably one, two orthree), optionally comprising an N-terminal protection group, or asdescribed in WO 2009/118375, WO 98/13459) or a protease inhibitor of theprotein type such as RASI, BASI, WASI (bifunctionalalpha-amylase/subtilisin inhibitors of rice, barley and wheat) or Cl2 orSSI. The composition may be formulated as described in, e.g., WO92/19709, WO 92/19708 and U.S. Pat. No. 6,472,364. In some embodiments,the enzymes employed herein are stabilized by the presence ofwater-soluble sources of zinc (II), calcium (II) and/or magnesium (II)ions in the finished compositions that provide such ions to the enzymes,as well as other metal ions (e.g., barium (II), scandium (II), iron(II), manganese (II), aluminum (III), Tin (II), cobalt (II), copper(II), Nickel (II), and oxovanadium (IV)).

In some preferred embodiments, the detergent compositions are typicallyformulated such that, during use in aqueous cleaning operations, thewash water has a pH of from about 5.0 to about 11.5, or in alternativeembodiments, even from about 6.0 to about 10.5. In some preferredembodiments, granular or liquid laundry products are formulated to havea pH from about 6 to about 8. Techniques for controlling pH atrecommended usage levels include the use of buffers, alkalis, acids,etc., and are well known to those skilled in the art.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1: Construction of Variants by Site-DirectedMutagenesis

Site-directed variants were constructed of subtilisin 309 (SEQ ID NO: 1)comprising specific substitutions according to the invention. Thevariants were made by traditional cloning of DNA fragments (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor, 1989) using PCR together with properly designed mutagenicoligonucleotides that introduced the desired mutations in the resultingsequence.

Mutagenic oligos were synthesized corresponding to the DNA sequenceflanking the desired site(s) of mutation, separated by the DNA basepairs defining the insertions/deletions/substitutions. In this manner,the variants listed in Table 1 below were constructed and produced.

In order to test subtilisin 309 variants of the invention, the mutatedDNA comprising a variant of the invention was transformed into acompetent B. subtilis strain, fermented using standard protocols (PS-1media, 3-4 days, 37° C.) and purified.

TABLE 1 Subtilisin 309 Variants Code Mutations A-000 Y167A + R170S +A194P A-001 Q59D + Y167A + R170S + A194P + L262E A-002 Q59D + N76D +Y167A + R170S + A194P A-003 N76D + Y167A + R170S + A194P + N238E A-004V104T + Y167A + R170S + A194P + Y209W + N238E + L262E A-005 V104T +H120D + S163G + Y167A + R170S + A194P + N261D A-006 V104T + S156D +Y167A + R170S + A194P + Y209W + L262E A-007 V104T + Y167A + R170S +A194P + N238E + L262E A-008 Q59D + Y167A + R170S + A194P + Y209W + L262EB-000 *99aE B-001 *99aE + N238E + L262E B-002 Q59D + *99aE + Y209W +L262E B-003 *99aE + B194P + G195E + L262E B-004 *99aE + S101H + H120D +S163G + N261D B-005 *99aE + S156D + Y209W + L262E B-006 *99aE + B194P +G195E + Y209W + L262E C-000 N62D + R170S C-001 N62D + S156D + S163G +Y209W + L262E C-002 N62D + R170S + Y209W + L262E C-003 N62D + R170S +N238E + L262E C-004 N62D + S156D + R170S + Y209W + L262E C-005 N62D +V104T + R170S + Y209W + L262E C-006 N62D + V104T + S156D + R170S +Y209W + L262E C-007 N62D + S101H + R170S + Y209W + L262E D-000 N62D +R170S + Q245R + N248D D-001 N62D + S156D + S163G + Y209W + Q245R +N248D + L262E D-002 N62D + R170S + Y209W + Q245R + N248D + L262E D-003N62D + S156D + S163K + Y209W + Q245R + N248D + L262E

Example 2: Wash Testing of Variants by Automatic Mechanical Stress Assay(AMSA)

Washing experiments were performed in order to assess the washperformance of selected protease variants in laundry detergent. Theproteases of the present application were tested using the AutomaticMechanical Stress Assay (AMSA). With the AMSA, the wash performance ofmany small volume enzyme-detergent solutions can be examined. The AMSAplate has a number of slots for test solutions and a lid that firmlysqueezes a textile swatch to be washed against the slot openings. Duringthe wash, the plate, test solutions, soiled textile swatch and lid arevigorously shaken to bring the test solution in contact with the soiledtextile swatch and apply mechanical stress in a regular, periodicoscillating manner. For further description see WO 02/42740 especiallythe paragraph “Special method embodiments” at pages 23-24.

Model detergent and test materials were as provided in Table 2A:

TABLE 2A Composition of model detergents and test materials Laundry 0.3to 0.5% xanthan gum, liquid 0.2 to 0.4% antifoaming agent, model 6 to 7%glycerol, 0.3 to 0.5% ethanol, detergent 4 to 7% FAEOS (fatty alcoholether sulfate), 24 to 28% nonionic surfactants, 1% boric acid, 1 to 2%sodium citrate (dihydrate), 2 to 4% soda, 14 to 16% coconut fatty acid,0.5% HEDP (1-hydroxyethane-(1,1-diphosphonic acid)), 0 to 0.4% PVP(polyvinylpyrrolidone), 0 to 0.05% optical brighteners, 0 to 0.001% dye,remainder deionized water.

The experiment was conducted under the experimental conditions asspecified in Table 2B below.

TABLE 2B AMSA Experimental Conditions Detergent Laundry liquid modeldetergent Detergent dosage 4.66 g/L Test solution volume 160 pL pH 7.6Wash time 20 minutes Temperature 20° C. Water hardness 16° dH Enzymeconcentration 8.0 mg enzyme protein/L in test solution Test materialChocolate milk and soot swatch (PC-03)

Water hardness was adjusted to 16° dH by addition of CaCl₂, MgCl₂, andNaHCO₃ (Ca²⁺:Mg²⁺:CO₃ ²⁻=5:1:6) to the test system. After washing thetextile swatches were flushed in tap water and dried.

The performance of an enzyme variant was measured as the brightness ofthe color of the textile washed with that specific protease. Brightnesscan also be expressed as the intensity of the light reflected from thesample when illuminated with white light. When the sample is stained theintensity of the reflected light is lower, than that of a clean sample.Therefore the intensity of the reflected light can be used to measurewash performance of a protease. Color measurements were made with aprofessional flatbed scanner (EPSON EXPRESSION 10000XL, Atea A/S,Lautrupvang 6, 2750 Ballerup, Denmark), which was used to capture animage of the washed melamine tiles.

To extract a value for the light intensity from the scanned images, aspecial designed software application was used (Novozymes Colour VectorAnalyzer). The program retrieves the 24 bit pixel values from the imageand converts them into values for red, green and blue (RGB). Theintensity value (Int) was calculated by adding the RGB values togetheras vectors and then taking the length of the resulting vector:

Int=√{square root over (r ² +g ² +b ²)}

Textiles

Standard chocolate milk and soot textile swatches (PC-03) were obtainedfrom the Center For Testmaterials BV, P.O. Box 120, 3133 KT Vlaardingen,the Netherlands.

The variants had a significantly better wash performance than the parentbackbone.

TABLE 3 AMSA wash performance data for variants relative to parentbackbone (A-000). AMSA wash performance (relative to parent CodeMutations backbone) A-001 Q59D + Y167A + R170S + 1.2 A194P + L262E A-002Q59D + N76D + Y167A + 1.1 R170S + A194P A-003 N76D + Y167A + R170S + 1.1A194P + N238E A-004 V104T + Y167A + R170S + A194P + 1.1 Y209W + N238E +L262E A-005 V104T + H120D + S163G + Y167A + 1.1 R170S + A194P + N261DA-006 V104T + S156D + Y167A + R170S + 1.1 A194P + Y209W + L262E A-007V104T + Y167A + R170S + A194P + 1.1 N238E + L262E A-008 Q59D + Y167A +R170S + A194P + 1.1 Y209W + L262E A-000 Y167A + R170S + A194P 1.0

TABLE 4 AMSA wash performance data for variants relative to parentbackbone (B-000). AMSA wash performance (relative to Code Mutationsparent backbone) B-001 *99aE + N238E + L262E 1.1 B-002 Q59D + *99aE +Y209W + L262E 1.1 B-003 *99aE + A194P + G195E + L262E 1.1 B-004 *99aE +S101H + H120D + S163G + N261D 1.1 B-005 *99aE + S156D + Y209W + L262E1.1 B-006 *99aE + A194P + G195E + Y209W + L262E 1.1 B-000 *99aE 1.0

TABLE 5 AMSA wash performance data for variants relative to parentbackbone (C-000). AMSA wash performance (relative to Code Mutationsparent backbone) C-001 N62D + S156D + S163G + Y209W + L262E 1.3 C-002N62D + R170S + Y209W + L262E 1.2 C-003 N62D + R170S + N238E + L262E 1.2C-004 N62D + S156D + R170S + Y209W + L262E 1.1 C-005 N62D + V104T +R170S + Y209W + L262E 1.1 C-006 N62D + V104T + S156D + R170S + 1.1Y209W + L262E C-007 N62D + S101H + R170S + Y209W + L262E 1.1 C-000N62D + R170S 1.0

TABLE 6 AMSA wash performance data for variants relative to parentbackbone (D-000). AMSA wash performance (relative to parent CodeMutations backbone) D-001 N62D + S156D + S163G + Y209W + 1.2 Q245R +N248D + L262E D-002 N62D + R170S + Y209W + Q245R + 1.1 N248D + L262ED-003 N62D + S156D + S163G + Y209W + 1.1 Q245R + N248D + L262E D-000N62D + R170S + Q245R + N248D 1.0

Example 3

The wash performance of variants in detergents was determined by usingthe following standardized stains obtainable from CFT (Center forTestmaterials) B.V., Vlaardingen, Netherlands:

A: chocolate-milk/ink on cotton: product no. C3B: chocolate-milk/ink on polyester/cotton: product no. PC3C: blood-milk/ink on cotton: product no. C5D: blood-milk/ink on polyester/cotton: product no. PC5E: peanut oil pigment/ink on cotton: product no. C10F: egg/pigment on cotton: product no. CS37

Furthermore the following stain obtainable from Eidgenössische Material-and Prüfanstalt (EMPA) was used:

G: grass on cotton: product no. 164

A liquid washing agent with the following composition was used as baseformulation (all values in weight percent): 0 to 0.5% xanthan gum, 0.2to 0.4% antifoaming agent, 0.2 to 8% Triethanolamine, 1 to 7% glycerol,0.3 to 3% ethanol, 0 to 12% FAEOS (fatty alcohol ether sulfate), 1 to28% nonionic surfactants, 0.5-4% boric acid, 0.5 to 6% sodium citrate(dihydrate), 1 to 6% soda, 0 to 16% coconut fatty acid, 0.5 to 6% HEDP(1-hydroxyethane-(1,1-diphosphonic acid)), 0 to 0.4% PVP(polyvinylpyrrolidone), 0 to 0.05% optical brighteners, 0 to 0.001% dye,remainder deionized water.

Based on this base formulation, various detergents were prepared byadding respective proteases as indicated in table 7a) and b). Referenceis the protease Subtilisin309 that has the amino acid sequence of SEQ IDNO. 1 the reference protease already showing a good wash performance,especially in liquid detergents. The proteases were added in the sameamounts based on total protein content (5 mg/I wash liquor).

The dosing ratio of the liquid washing agent was 4.7 grams per liter ofwashing liquor and the washing procedure was performed for 60 minutes ata temperature of 20° C. and 40° C., the water having a water hardnessbetween 15.5 and 16.5° (German degrees of hardness).

The whiteness, i.e. the brightening of the stains, was determinedphotometrically as an indication of wash performance. A Minolta CM508dspectrometer device was used, which was calibrated beforehand using awhite standard provided with the unit.

The results obtained are the difference values between the remissionunits obtained with the detergents and the remission units obtained withthe detergent containing the reference protease. A positive valuetherefore indicates an improved wash performance of the variants in thedetergent. It is evident from tables 7a (results at 40° C.) and 7b(results at 20° C.) that variants according to the invention showimproved wash performance.

TABLE 7a Wash performance at 40° C. of protease variants that have thesame amino acid sequence as SEQ ID NO: 1 except for the substitutions asper the table below on the stains as indicated; reference is theprotease according to SEQ ID NO: 1. A B C D E F G *99aE, A194P nd nd 5.4nd nd  8.3 1.7 N76D, Y167A, R170S, A194P 1.5 nd 2.8 nd 0.5 nd 0.6 N76D,Y167A, R170S, A194P, 2.4 nd 3.0 nd 1.0 nd nd A228V, A230V *99aE, S256D1.5 0.9 4.1 6.6 nd  4.4 nd L21D, *99aE nd 0.8 5.2 6.7 0.7  5.0 nd N62D,Q245R, R170S 0.6 0.7 5.5 7.7 0.5  1.5 0.5 R170L, Q206E, S57P 2.0 0.9 4.96.3 1.2  1.2 0.9 A133P, Y167A, R170S, A194P nd nd 3.5 5.5 1.1 nd 1.3S141N, Y167A, R170S, A194P 1.4 0.8 5.1 5.5 nd nd 1.3 Y167A, R170N nd 0.84.7 4.9 nd  0.5 1.6 Y167A, R170S, A172E nd 0.6 1.7 5.3 nd  1.9 nd N62D,Y167A, R170S, A194P nd 0.8 3.0 6.6 nd 11.6 nd N62D, R170S nd 0.6 4.3 7.4nd  7.4 nd N62D, R170L 0.6 nd 3.1 8.4 nd  9.4 nd *97aN, A98T, S99D 0.6nd 3.4 7.3 nd  5.5 nd *98aA, S99D, N261D, L262Q nd nd 4.3 5.8 nd  6.9 nd

TABLE 7b Wash performance at 20° C. of protease variants that have thesame amino acid sequence as SEQ ID NO: 1 except for the substitutions asper the table below on the stains as indicated; reference is theprotease according to SEQ ID NO: 1. A B C D E F G *99aE, A194P 0.5 nd3.1 nd 2.0 3.4 0.6 N76D, Y167A, R170S, A194P 2.0 3.5 2.6 3.6 0.5 2.2 ndN76D, Y167A, R170S, A194P, 2.9 3.3 2.7 4.5 1.0 0.8 nd A228V, A230V*99aE, S256D 2.3 2.4 1.9 4.0 1.6 4.6 nd L21D, *99aE nd 1.8 nd 4.4 2.24.7 nd N62D, Q245R, R170S 2.8 1.0 3.1 4.9 nd 1.1 nd R170L, Q206E, S57P2.5 1.8 2.3 2.9 1.8 nd nd A133P, Y167A, R170S, A194P 2.1 1.6 1.7 3.5 2.0nd 0.8 S141N, Y167A, R170S, A194P nd nd nd 3.3 2.3 1.0 0.7 Y167A, R170Nnd 0.6 nd 3.0 2.4 nd 1.2 Y167A, R170S, A172E 3.1 2.7 0.5 2.4 1.9 nd ndN62D, Y167A, R170S, A194P 2.8 5.6 2.7 4.3 2.0 2.5 nd N62D, R170S 2.1 0.51.9 4.6 1.0 0.7 nd N62D, R170L 3.5 4.0 1.6 4.6 nd nd nd *97aN, A98T,S99D nd nd 2.2 4.2 1.5 4.8 1.1 *98aA, S99D, N261D, L262Q nd nd 2.8 3.5nd 4.9 nd

1. A subtilase variant comprising a set of alterations selected from the group consisting of: (a) X167A+R170S+A194P and one or more substitutions selected from the group consisting of X59D, X62D, X76D, X104T, X120D, X133P, X141N, X156D, X163G, X209W, X228V, X230V, X238E, X261D, and X262E; (b) *99aE and one or more substitutions selected from the group consisting of X21D, X59D, X101H, X120D, X156D, X163G, X194P, X195E, X209W, X238E, X256D, X261D, and X262E; (c) X62D and one or more substitutions selected from the group consisting of X101H, X104T, X156D, X163G, X170S, X170L, X209W, X238E, X245R and X262E; (d) X62D+X245R+X248D and one or more substitutions selected from the group consisting of X156D, X163G, X163K, X170S, X209W, and X262E; (e) X170L, X170N or X170S and one or more substitutions selected from the group consisting of X57P, X167A, X172E, X206E, (f) X99D and one or more substitutions selected from the group consisting of *97aN, *98aA, X98T, X261D, and X262Q, wherein (i) the positions correspond to the positions of the polypeptide of SEQ ID NO: 2; (ii) the variant has protease activity; and (iii) the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 or
 2. 2. The subtilase variant according to claim 1, which comprises the substitutions X167A+X170S+X194P and one or more substitutions selected from the group consisting of X59D, X62D, X76D, X104T, X120D, X133P, X141N, X156D, X163G, X209W, X228V, X230V, X238E, X261D, and X262E.
 3. The subtilase variant according to claim 1, which comprises the alteration *99aE and one or more substitutions selected from the group consisting of X21D, X59D, X101H, X120D, X156D, X163G, X194P, X195E, X209W, X238E, X256D, X261D, and X262E.
 4. The subtilase variant according to claim 1, which comprises the substitution X62D and one or more substitutions selected from the group consisting of X101H, X104T, X156D, X163G, X170S, X170L, X209W, X238E, X245R and X262E.
 5. The subtilase variant according to claim 1, which comprises the substitutions X62D+X245R+X248D and one or more substitutions selected from the group consisting of X156D, X163G, X163K, X170S, X209W, and X262E.
 6. The subtilase variant according to claim 1, which comprises the substitutions X170L, X170N or X170S and one or more substitutions selected from the group consisting of X57P, X167A, X172E, X206E.
 7. The subtilase variant according to claim 1, which comprises the substitution X99D and one or more alterations selected from the group consisting of *97aN, *98aA, X261D, and X262Q.
 8. The subtilase variant according to any of claim 1, which further comprises one or more alterations selected from the group consisting of X3T, X4I, X9C, X9D, X9E, X9Q, X14T, X24G, X24R, X27R,*36D, X43A, X43C, X43L, X43R, X43W, X68A, X72A, X72V, X76D, X78D, X87R, X87S,*97E, X98S, X99A, X99D, X99A, X99D, X99E, X99G,*99aD, X101D, X101E, X101G, X101I, X101K, X101L, X101M, X101N, X101R, X103A, X104F, X104I, X104N, X104Y, X106A, X114V, X115T, X115W, X118R, X118V, X120D, X120I, X120N, X120T, X120V, X123S, X128A, X128L, X128S, X129D, X129N, X129Q, X130A, X147W, X149C, X149N, X158E, X160D, X160P, X161C, X161E, X162L, X163A, X163D, X182C, X182E, X185C, X185E, X188C, X188D, X188E, X191N, X195E, X199M, X204D, X204V, X205I, X206C, X206E, X206I, X206K, X206L, X206T, X206V, X206W, X209W, X212A, X212D, X212G, X212N, X216I, X216T, X216V, X217C, X217D, X217E, X217M, X217Q, X217Y, X218D, X218E, X218T, X222C, X222R, X222S, X225A, X232V, X235L, X236H, X245K, X245R, X252K, X255C, X255E, X256A, X256C, X256D, X256V X256Y, X259D, X260E, X260P, X261C, X261E, X261F, X261L, X261M, X261V, X261W, X261Y, X262C, X262E, X262Q, and X274A, wherein each position corresponds to the position of the polypeptide of SEQ ID NO:
 2. 9. The subtilase variant according to claim 1, comprising or consisting of a set of alterations selected from the group consisting of: *99aE+A194P N76D+Y167A+R170S+A194P N76D+Y167A+R170S+A194P+A228V+A230V 99aE+S256D L21D+*99aE N62D+Q245R+R170S R170L+Q206E+S57P A133P+Y167A+R170S+A194P S141N+Y167A+R170S+A194P Y167A+R170N Y167A+R170S+A172E N62D+Y167A+R170S+A194P N62D+R170S N62D+R170L 97aN+A98T+S99D 98aA+S99D+N261D+L262Q Q59D+N76D+Y167A+R170S+A194P; Q59D+*99aE+Y209W+L262E; Q59D+Y167A+R170S+A194P+Y209W+L262E; Q59D+Y167A+R170S+A194P+L262E; N62D+S101H+R170S+Y209W+L262E; N62D+V104T+S156D+R170S+Y209W+L262E; N62D+V104T+R170S+Y209W+L262E; N62D+S156D+S163G+Y209W+Q245R+N248D+L262E; N62D+S156D+S163G+Y209W+L262E; N62D+S156D+S163K+Y209W+Q245R+N248D+L262E; N62D+S156D+R170S+Y209W+L262E; N62D+R170S+Y209W+Q245R+N248D+L262E; N62D+R170S+Y209W+L262E; N62D+R170S+N238E+L262E; N76D+Y167A+R170S+A194P+N238E; *99aE+S101H+H120D+S163G+N261D; *99aE+S156D+Y209W+L262E; *99aE+B194P+G195E+Y209W+L262E; *99aE+B194P+G195E+L262E; *99aE+N238E+L262E; V104T+H120D+S163G+Y167A+R170S+A194P+N261D; V104T+S156D+Y167A+R170S+A194P+Y209W+L262E; V104T+Y167A+R170S+A194P+Y209W+N238E+L262E; and V104T+Y167A+R170S+A194P+N238E+L262E.
 10. The subtilase variant according to claim 1, which is a variant of subtilisin 309 (SEQ ID NO: 1), comprising or consisting of the set of alterations.
 11. The subtilase variant according to claim 1, which is a variant of subtilisin BPN′ (SEQ ID NO: 2), comprising or consisting of the set of alterations.
 12. The subtilase variant according to claim 1, which has an improved wash performance compared to SEQ ID NO: 1 when measured in AMSA assay.
 13. The subtilase variant according to claim 1, wherein the total number of alterations compared to SEQ ID NO: 1 is between 3 and
 30. 14. A method for producing a subtilase variant of claim 1, comprising (a) introducing into a parent subtilase a set of alterations selected from the group consisting of: (1) X167A+R170S+A194P and one or more substitutions selected from the group consisting of X59D, X62D, X76D, X104T, X120D, X133P, X141N, X156D, X163G, X209W, X228V, X230V, X238E, X261D, and X262E; (2) *99aE and one or more substitutions selected from the group consisting of X21D, X59D, X101H, X120D, X156D, X163G, X194P, X195E, X209W, X238E, X256D, X261D, and X262E; (3) X62D and one or more substitutions selected from the group consisting of X101H, X104T, X156D, X163G, X170S, X170L, X209W, X238E, X245R and X262E; (4) X62D+X245R+X248D and one or more substitutions selected from the group consisting of X156D, X163G, X163K, X170S, X209W, and X262E; (5) X170L, X170N or X170S and one or more substitutions selected from the group consisting of X57P, X167A, X172E, X206E, (6) X99D and one or more substitutions selected from the group consisting of *97aN, *98aA, X98T, X261D, and X262Q; wherein (i) the positions correspond to the positions of the polypeptide of SEQ ID NO: 2; (ii) the variant has protease activity; and (iii) the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 or
 2. (b) recovering the variant.
 15. The subtilase variant of claim 8, which comprises Y209W.
 16. The subtilase variant of claim 8, which comprises L262E. 